Method and means to modulate programmed cell death in eukaryotic cells

ABSTRACT

The invention provides for the use of isolated polynucleotides encoding maize poly (ADP-ribose) polymerase (PARP) proteins to produce eukaryotic cells and organisms, particularly plant cells and plants, with modified programmed cell death. Eukaryotic cells and organisms particularly plant cells and plants, are provided wherein either in at least part of the cells, preferably selected cells, the programmed cell death (PCD) is provoked, or wherein, on the contrary, PCD of the cells or of at least part of the cells in an organism is inhibited, by modulation of the level or activity or PARP proteins in those cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/705,197, filed Nov. 12, 2003, now U.S. Pat. No. 7,241,936, which is acontinuation of U.S. patent application Ser. No. 09/118,276, filed Jul.17, 1998, now U.S. Pat. No. 6,693,185, the disclosures of each of whichare hereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to the use of poly (ADP-ribose) polymerase (PARP)proteins, particularly mutant PARP proteins or parts thereof, and genesencoding the same, to produce eukaryotic cells and organisms,particularly plant cells and plants, with modified programmed celldeath. Eukaryotic cells and organism, particularly plant cells andplants, are provided wherein either in at least part of the cells,preferably selected cells, the programmed cell death (PCD) is provoked,or wherein, on the contrary, PCD of the cells or of at least part of thecells in an organism is inhibited, by modulation of the level oractivity of PARP proteins in those cells. The invention also relates toeukaryotic cells and organisms, particularly plant cells and plants,expressing such genes.

DESCRIPTION OF RELATED ART

Programmed cell death (PCD) is a physiological cell death processinvolved in the elimination of selected cells both in animals and inplants during developmental processes or in response to environmentalcues (for a review see Ellis et al. 1991; Pennell and Lamb, 1997). Thedisassembly of cells undergoing PCD is morphologically accompanied bycondensation, shrinkage and fragmentation of the cytoplasm and nucleus,often into small sealed packets (Cohen 1993, Wang at al. 1996).Biochemically, PCD is characterized by fragmentation of the nuclear DNAinto generally about 50 kb fragments representing oligonucleosomes, aswell as the induction of cysteine proteinases and endonucleases. Thefragmentation of the DNA can be detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) of DNA 3′-OH groupsin sections of cells. (Gavrieli et al. 1992). Cell death by PCD isclearly distinct from cell death by necrosis, the latter involving cellswelling, lysis and leakage of the cell contents.

In animals, PCD is involved in the elimination or death of unwantedcells such as tadpole tail cells at metamorphosis, cells betweendeveloping digits in vertebrates, overproduced vertebrate neurons, cellsduring cell specialization such as keratocytes etc. Damaged cells, whichare no longer able to function properly, can also be eliminated by PCD,preventing them from multiplying and/or spreading. PCD, or the lackthereof, has also been involved in a number of pathological conditionsin humans (AIDS, Alzheimer's disease, Huntington's disease, LouGehring's disease, cancers).

In plants, PCD has been demonstrated or is believed to be involved in anumber of developmental processes such as e.g., removal of the suspensorcells during the development of an embryo, the elimination of aleuronecells after germination of monocotyledonous seeds; the elimination ofthe root cap cells after seed germination and seedling growth; celldeath during cell specialization as seen in development of xylemtracheary element or trichomes, or floral organ aborting in, unisexualflowers. Also the formation of aerochyma in roots under hypoxicconditions and the formation of leaf lobes or perforations in someplants seem to involve PCD. Large scale cell death in plants occursduring upon senescence of leaves or other organs. The hypersensitiveresponse in plants, in other words the rapid cell death occurring at thesite of entry of an avirulent pathogen leading to a restricted lesion,is an another example of PCD in response to an environmental cue.

Animal or plant cells dying in suspension cultures, particularly inlow-density cell suspension cultures, also demonstrate thecharacteristics of PCD.

An enzyme which has been implied to be involved in PCD or apoptosis ispoly(ADP-ribose) polymerase. Poly(ADP-ribose) polymerase (PARP), alsoknown as poly(ADP-ribose) transferase (ADPRT) (EC 2.4.2.30), is anuclear enzyme found in most eukaryotes, including vertebrates,arthropods, molluscs, slime moulds, dinoflagellates, fungi and other loweukaryotes with the exception of yeast. The enzymatic activity has alsobeen demonstrated in a number of plants (Payne et al., 1976; Willmitzerand Wagner, 1982; Chen et al., 1994; O'Farrell, 1995).

PARP catalyzes the transfer of an ADP-ribose moiety derived from NAD⁺,mainly to the carboxyl group of a glutamic acid residue in the targetprotein, and subsequent ADP-ribose polymerization. The major targetprotein is PARP itself, but also histones, high mobility groupchromosomal proteins, a topoisomerase, endonucleases and DNA polymeraseshave been shown to be subject to this modification.

The PARP protein from animals is a nuclear protein of 113-120 kDa,abundant in most cell types, that consist of three major functionaldomains: an amino-terminal DNA-binding domain containing two Zn-fingerdomains, a carboxy-terminal catalytic domain, and an internal domainwhich is automodified (de Murcia and Ménissier de Murcia, 1994;Kameshita et al., 1984; Lindahl et al., 1995). The enzymatic activity invitro is greatly increased upon binding to single-strand breaks in DNA.The in vivo activity is induced by conditions that eventually result inDNA breaks (Alvarez-Gonzalez and Althaus, 1989; Ikejima et al., 1990).Automodification of the central domain apparently serves as a negativefeedback regulation of PARP.

PARP activity in plant cells was first demonstrated by examining theincorporation of ³H from labelled NAD⁺ into the nuclei of root tip cells(Payne et al., 1976; Willmitzer and Wagner, 1982). The enzymaticactivity was also partially purified from maize seedlings and found tobe associated with a protein of an apparent molecular mass of 113 kDa,suggesting that the plant PARP might be similar to the enzyme fromanimals (Chen et al., 1994; O'Farrell, 1995).

cDNAs corresponding to PARP proteins have isolated from several speciesincluding mammals, chicken, Xenopus, insects and Caenorhabditis elegans.

Chen et al. (1994) have reported PARP activity in maize nuclei andassociated this enzymatic activity with the presence of an approximately114 kDa protein present in an extract of maize nuclei. O'Farrel (1995)reported that RT-PCR-amplification on RNA isolated from maize (usingdegenerate primers based on the most highly conserved sequences)resulted in a 300 bp fragment, showing 60% identity at the amino acidlevel with the human PARP protein. Lepiniec et al (1995) have isolatedand cloned a full length cDNA from Arabidopsis thaliana encoding a 72kDa protein with high similarity to the catalytic domain of vertebratePARP. The N-terminal domain of the protein does not reveal any sequencesimilarity with the corresponding domain of PARP from vertebrates but iscomposed of four stretches of amino acids (named A1, A2, B and C)showing similarity to the N-terminus of a number of nuclear and DNAbinding proteins. The predicted secondary structure of A1 and A2 was ahelix-loop-helix structure.

The Genbank database contains the sequences of two cDNAS from Zea maysfor which the amino acid sequence of the translation products has eitherhomology to the conventional PARP proteins (AJ222589) or to thenon-conventional PARP proteins, as identified in Arabidopsis (AJ222588)

The function(s) of PARP and poly-ADP ribosylation in eukaryotic cells is(are) not completely clear. PARP is involved or believed to be involvedeither directly or indirectly in a number of cellular processes such asDNA repair, replication and recombination, in cell division and celldifferentiation or in the signalling pathways that sense alterations inthe integrity of the genome. As PARP activity may significantly reducethe cellular NAD⁺ pool, it has also been suggested that the enzyme mayplay a critical role in programmed cell death (Heller et al., 1995;Zhang et al., 1994). Further, it has been suggested that nicotinamideresulting from NAD⁺ hydrolysis or the products of the turn-over ofpoly-ADP-ribose by poly-ADP-ribose glycohydrolase may be stress responsesignals in eukaryotes.

The information currently available on the biological function of plantPARP has come from experiments involving PARP inhibitors suggesting anin vivo role in the prevention of homologous recombination at sites ofDNA damage as rates of homologous intrachromosomal recombination intobacco are increased after application of 3-aminobenzamide (3ABA)(Puchta et al., 1995). Furthermore, application of PARP inhibitors, suchas 3ABA, nicotinamide, and 6(5H)-phenasthridinone, to differentiatingcells of Zinnia or of Helianthus tuberosum has been shown to preventdevelopment of tracheary elements (Hawkins and Phillips, 1983; Phillipsand Hawkins, 1985; Shoji et al., 1997; Sugiyama et al., 1995), which isconsidered to be an example of programmed cell death in plants.

PCT application WO97/06267 describes the use of PARP inhibitors toimprove the transformation (qualitatively or quantitatively) ofeukaryotic cells, particularly plant cells.

Lazebnik et al. (1994) identified a protease with properties similar tothe interleukin 1-β-converting enzyme capable of cleaving PARP, which isan early event in apoptosis of animal cells.

Kuepper et al. (1990) and Molinette et al. (1993) have described theoverproduction of the 46 kDa human PARP DNA-binding domain and variousmutant forms thereof, in transfected CV-1 monkey cells or humanfibroblasts and have demonstrated the trans-dominant inhibition ofresident PARP activity and the consequent block of base excision DNArepair in these cells.

Ding et al. (1992), and Smulson et al. (1995) have described depletionof PARP by antisense RNA expression in mammalian cells and observed adelay in DNA strand break joining, and inhibition of differentiation of3T3-L1 preadipocytes.

Ménissier de Murcia et al., (1997) and Wang et al. (1995, 1997) havegenerated transgenic “knock-out” mice mutated in the PARP gene,indicating that PARP is not an essential protein. Cells ofPARP-deficient mice are, however, more sensitive to DNA damage anddiffer from normal cells of animals in some aspects of induced celldeath (Heller et al., 1995).

SUMMARY AND OBJECTS OF THE INVENTION

The invention provides a method for modulating programmed cell death ina eukaryotic cell, comprising reducing the functional level of the totalPARP activity in a eukaryotic cell using the nucleotide sequence of aPARP gene of the ZAP class, and the nucleotide sequence of a PARP geneof the NAP class, preferably to reduce expression of the endogeneousPARP genes, to reduce the apparent activity of the proteins encoded bythe endogenous PARP genes or to alter the nucleotide sequence of theendogenous PARP genes.

The invention also provides a method for modulating programmed celldeath in a eukaryotic cell, comprising introducing a first and a secondPCD modulating chimeric gene in a eukaryotic cell, preferably a plantcell, wherein the first PCD modulating chimeric gene comprises thefollowing operably linked DNA regions: a promoter, operative in aeukaryotic cell; a DNA region, which when transcribed yields a RNAmolecule which is either capable of reducing the functional level of aZn-finger containing PARP protein of the ZAP class; or is capable ofbeing translated into a peptide or protein which when expressed reducesthe functional level of a PARP protein of ZAP class and a DNA regioninvolved in transcription termination and polyadenylation

and wherein the second PCD modulating chimeric gene comprises thefollowing operably linked DNA regions: a promoter, operative in theeukaryotic cell; a DNA region, which when transcribed yields a RNAmolecule which is either capable of reducing the functional level of aPARP protein of the NAP class; or capable of being translated into apeptide or protein which when expressed reduces the functional level ofa PARP protein of the NAP class, and a DNA region involved intranscription termination and polyadenylation; and wherein the totalapparent PARP activity in the eukaryotic cell is reduced significantly,(preferably the total apparent PARP activity is reduced from about 75%to about 90% of the normal apparent PARP activity in the eukaryoticcell, and the eukaryotic cell is protected against programmed celldeath) or almost completely (preferably the total apparent PARP activityis reduced from about 90% to about 100% of the normal apparent PARPactivity in the eukaryotic cell, and the cell is killed by programmedcell death).

Preferably the first transcribed DNA region or the second transcribedDNA region or both, comprise a nucleotide sequence of at least about 100nucleotides with 75% identity to the sense DNA strand of an endogenousPARP gene of the ZAP or the NAP class, and encode a sense RNA moleculeis capable of reducing the expression of the endogenous PARP gene of theZAP or the NAP class.

In an alternative method for modulating programmed cell death, providedby the invention, the first transcribed DNA region or the secondtranscribed DNA region or both, comprise a nucleotide sequence of atleast about 100 nucleotides with 75% identity to the complement of thesense DNA strand of an endogenous PARP gene of the ZAP or the NAP class,and encode RNA molecule is capable of reducing the expression of saidendogenous PARP gene of the ZAP or the NAP class.

In yet an alternative method for modulating programmed cell death,provided by the invention, the first and/or second transcribed DNAregion encodes a RNA molecule comprising a sense nucleotide sequence ofat least about 100 nucleotides with 75% identity to the mRNA resultingfrom transcription of an endogenous PARP gene of the ZAP or the NAPclass and the RNA molecule further comprising an antisense nucleotidesequence of at least about 100 nucleotides with 75% identity to thecomplement of the mRNA resulting from transcription of the endogenousPARP gene of the ZAP or the NAP class, wherein the sense and antisensenucleotide sequence are capable of forming a double stranded RNA region,and wherein that RNA molecule is capable of reducing the expression ofthe endogenous PARP gene of the ZAP or the NAP class.

In a further alternative method for modulating programmed cell death,provided by the invention, the first and/or second transcribed DNAregion encodes a dominant negative PARP mutant capable of reducing theapparent activity of the PARP protein encoded by an endogenous PARP geneof the ZAP or the NAP class, preferably comprising amino acid sequenceselected from the amino acid sequence of SEQ ID No 4 from amino acid 1to 159 or the amino acid sequence of SEQ ID No 6 from amino acid 1 to138 or comprising an amino acid sequence selected from the amino acidsequence of SEQ ID No 2 from amino acid 1 to 370, the amino acidsequence of SEQ ID No 11 from amino acid 1 to 98, or the amino acidsequence of SEQ ID No 2 from amino acid 1 to 370 wherein the amino acidsequence from amino acid 1 to 88 is replaced by the amino acid sequenceof SEQ ID No 11.

The promoter of the first and second chimeric PCD modulating genes, orboth, may be a tissue specific or inducible promoter such as a promoteris selected from a fungus-responsive promoter, a nematode-responsivepromoter, an anther-selective promoter, a stigma-selective promoter, adehiscence-zone selective promoter.

The invention also provides a method for modulating programmed celldeath in a plant cell, comprising introduction of a PCD modulatingchimeric gene in said plant cell, wherein the PCD modulating chimericgene comprises the following operably linked DNA regions: aplant-expressible promoter, a DNA region, which when transcribed yieldsa RNA molecule, which is either capable of reducing the expression ofendogenous PARP genes; or is capable of being translated into a peptideor protein which when expressed reduces the apparent PARP activity inthe plant cell, and a DNA region involved in transcription terminationand polyadenylation, wherein the total apparent PARP activity in theplant cell is reduced from about 75% to about 100% of the normalapparent PARP activity in the plant cell.

It is another objective of the invention to provide the first and secondchimeric PCD modulating gene as well as a eucaryotic cell, particularlya plant cell comprising the first and second chimeric PCD modulatinggene and non-human eukaryotic organisms, particularly plants comprisingsuch cells.

Finally, the invention also provides an isolated DNA sequence comprisingthe nucleotide sequence of SEQ ID No 1 from the nucleotide at position113 to the nucleotide at position 3022, an isolated DNA sequencecomprising the nucleotide sequence of SEQ ID No 10 from the nucleotideat position 81 to the nucleotide at position 3020 and an isolated DNAsequence comprising the nucleotide sequence of SEQ ID No 3 from thenucleotide at position 107 to the nucleotide at position 2068.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The deduced N-terminal amino acid sequences of plantpoly(ADP-ribose) polymerases.

-   (A) Alignment of the sequences upstream of the NAD⁺-binding domain    found in Arabidopsis thaliana APP (A.th. APP; EMBL accession number    Z48243; SEQ ID No 6) and the maize homolog NAP (Z.m. NAP; EMBL    accession number AJ222588; SEQ ID No 4). The domain division shown    is as previously proposed (Lepiniec et al., 1995). The nuclear    localization signal (NLS) located in the B domain is indicated by    the bracket. The sequence of the B domain is not very well conserved    between dicotyledonous and monocotyledonous plants. The C domain is    probably comparable in function to the automodification domain of    PARP from animals. The imperfect repeats, A1 and A2, are also    present in maize NAP. To illustrate the internally imperfect    two-fold symmetry within the repeat sequence, the properties of    amino acid residues are highlighted below the sequences as follows:    filled-in circles, hydrophobic residue; open circle, glycine; (+),    positively charged residue; (−), negatively charged residue; wavy    line, any residue. The axis of symmetry is indicated by the vertical    arrowhead and arrowhead lines mark the regions with the inverted    repetition of amino acid side chain properties.-   (B) Alignment of the DNA-binding and auto-catalytic domains of mouse    PARP and maize ZAP. Zn-finger-containing maize ZAP1 and ZAP2    (partial cDNA found by the 5′RACE PCR analysis) are indicated as    Z.m. ZAP (EMBL accession number AJ222589; SEQ ID No 2) and Z.m. ZAP    (race) (SEQ ID No 11 from amino acid at position 1 to amino acid at    position 98), respectively, and the mouse PARP, M.m. ADPRT    (Swissprot accession number P11103). The Zn-fingers and bipartite    NLS of the mouse enzyme are indicated by brackets, the Caspase 3    cleavage site by the asterisk, and the putative NLS in the ZAP    protein by the bracket in bold below the maize sequence. The amino    acid residues that are conserved in all sequences are boxed; amino    acid residues with similar physico-chemical properties are shaded    with the uppermost sequence as a reference.

FIG. 2. Comparison of the NAD+-binding domain of mouse PARP and plantPARP proteins. The range of the “PARP signature” is indicated above thesequences. Names and sequence alignment are as in FIG. 1.

FIG. 3. Estimation of the gene copy number and transcript size for thenap and zap genes.

(A) and (B) Maize genomic DNA of variety LG2080 digested with theindicated restriction endonucleases, resolved by agarose gelelectrophoresis, blotted, and hybridized with radioactively labelled DNAprobes prepared from the 5′ domains of the nap and zap cDNA, which donot encode the NAD⁺-binding domain. The hybridization pattern obtainedwith the nap probe (A) is simple and indicates a single nap gene in: themaize genome. As can be seen from the hybridization pattern (B), theremight be at least two zap genes. To determine the size of thetranscripts encoded by the zap and nap genes, approximately 1 μg ofpoly(A)⁺ RNA extracted from roots (lane 1) and shoots (lane 2) of6-day-old seedlings were resolved on an agarose gel after denaturationwith glyoxal, blotted, and hybridized with nap (C) and zap (D)³²P-labelled cDNA. ³³P 5′ end-labelled BstEII fragments of λDNA wereused as a molecular weight markers in both DNA and RNA gel blotexperiments; their positions are indicated in kb to the left of eachpanel.

FIG. 4. Analysis of APP expression in yeast.

-   (A) Schematic drawing of the expression cassette in pV8SPA. The    expression of the app cDNA is driven by a chimeric yeast promoter,    which consists of the minimal TATA box-containing promoter region of    the cycl gene (CYCL) and an upstream activating promoter region of    the ga110 gene (GAL10), the latter providing promoter activation by    galactose. Downstream regulatory sequences are derived from the gene    encoding phosphoglycerol kinase (3PGK) (Kuge and Jones, 1994). The    app-coding region is drawn with a division in putative domains as    proposed earlier (Lepiniec et al., 1995): A1 and A2 correspond to    imperfect 27-amino acid repeats, in between which there is a    sequence (B domain), rich in positively charged amino acids and    resembling the DNA-binding domains of a number of DNA-binding    proteins. The amino acid sequence of the B domain is shown below the    map and the stretch of arginine and lysine residues, which may    function as an NLS is drawn in bold. Methionine residues (M¹, M⁷²),    which may function as translation initiation codons, are indicated    above the map. The C domain is rich in glutamic acid residues,    resembling in its composition, but not in its sequence, the    auto-modification domain of PARP from animals.-   (B) Immunoblot (Western blot) and Northern blot analyses of the DY    (pYeDP1/8-2) and DY(pV8SPA) strains, indicated as (vector) and    (app), respectively. Strains were grown in SDC medium supplemented    with glucose (GLU), galactose (GAL), galactose and 3 mM of 3ABA    (GAL+3ABA), or galactose and 5 mM nicotinamide (GAL+NIC). Total RNA    or total protein were extracted from the same cultures. Ten    micrograms of total protein were fractionated by electrophoresis on    10% SDS-PAGE, electroblotted, and probed with anti-APP antisera.    Five micrograms of total RNA were resolved by electrophoresis on an    1.5% agarose gel, blotted onto nylon membranes, and hybridized with    ³²P-labeled DNA fragments derived from the app cDNA. Positions of    the molecular weight marker bands are indicated to the left in    kilobases (kb) and kilodalton (kDa).

FIG. 5. Poly(ADP-ribose) polymerase activity of the APP protein.

-   (A) The total protein extracts were prepared from DY(pYeDP1/8-2)    grown on SDC with 2% galactose (vector GAL) and DY(pV8SPA) grown    either on SDC with 2% glucose (app GLU), on SDC with 2% galactose    (app GAL), or on SDC with 2% galactose and 3 mM 3ABA (app GAL+3ABA).    To detect the synthesis of the poly(ADP-ribose) in these extracts,    samples were incubated with ³²P-NAD⁺ for 40 min at room temperature.    Two control reactions were performed: 100 ng of the purified human    PARP were incubated either in a reaction buffer alone (PARP) (lane    5), or with protein extract made from DY(pYeDP1/8-2) culture grown    on glucose (vector GLU+PARP) (lane 6). The autoradiograph obtained    after exposure of the dried gel to X-Omat Kodak film is shown. ORi    corresponds to the beginning of the sequencing gel.-   (B) Stimulation of poly(ADP-ribose) synthesis by DNA in protein    extracts from DY(pV8SPA). Amounts of sonicated salmon sperm DNA    added to the nucleic acid depleted yeast extracts are indicated in    μg ml⁻¹. The synthesis of the poly(ADP-ribose) is blocked by 3ABA,    which was added in one of the reactions at a concentration of 3 mM    (lane 5). To ensure the maximal recovery of the poly(ADP-ribose), 20    μg of glycogen were included as a carrier during precipitation    steps; this, as can be seen, however resulted in high carry-over of    the unincorporated label.

FIG. 6. Schematic representation of the T-DNA vectors comprising the PCDmodulating chimeric genes of the invention. P35S: CaMV35S promoter; L:cab22 leader; ZAP; coding region of a PARP gene of the ZAP class; 5′AAP:N-terminal part of the coding regon of a PARP gene of the ZAP class ininverted orientation; 3′ 35S: CaMV35S 3′ end transcription terminationsignal and polyadenylation signal; pACT2: promoter region of the actingene; pNOS; nopaline synthase gene promoter; gat: gentamycin acetyltransferase; bar: phosphinotricin acetyl transferase; 3′NOS: 3′ endtranscription termination signal and polyadenylation signal of nopalinesynthase gene; APP: coding region of a PARP gene of the NAP class;5′APP: N-terminal part of the coding regon of a PARP gene of the NAPclass in inverted orientation; LB: left T-DNA border; RB: right T-DNAborder; pTA29: tapetum specific promoter, pNTP303: pollen specificpromoter.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

For the purpose of the invention, the term “plant-expressible promoter”means a promoter which is capable of driving transcription in a plantcell. This includes any promoter of plant origin, but also any promoterof non-plant origin which is capable of directing transcription in aplant cell, e.g., certain promoters of viral or bacterial origin such asthe CaMV35S or the T-DNA gene promoters.

The term “expression of a gene” refers to the process wherein a DNAregion under control of regulatory regions, particularly the promoter,is transcribed into an RNA which is biologically active i.e., which iseither capable of interaction with another nucleic acid or protein orwhich is capable of being translated into a biologically activepolypeptide or protein. A gene is said to encode an RNA when the endproduct of the expression of the gene is biologically active RNA, suchas e.g. an antisense RNA or a ribozyme. A gene is said to encode aprotein when the end product of the expression of the gene is abiologically active protein or polypeptide.

The term “gene” means any DNA fragment comprising a DNA region (the“transcribed DNA region”) that is transcribed into a RNA molecule (e.g.,a mRNA) in a cell under control of suitable regulatory regions, e.g., aplant-expressible promoter. A gene may thus comprise several operablylinked DNA fragments such as a promoter, a 5′ leader sequence, a codingregion, and a 3′ region comprising a polyadenylation site. An endogenousplant gene is a gene which is naturally found in a plant species. Achimeric gene is any gene which is not normally found in a plant speciesor, alternatively, any gene in which the promoter is not associated innature with part or all of the transcribed DNA region or with at leastone other regulatory regions of the gene.

As used herein “comprising” is to be interpreted as specifying thepresence of the stated features, integers, steps or components asreferred to, but does not preclude the presence or addition of one ormore features, integers, steps or components, or groups thereof. Thus,e.g., a nucleic acid or protein comprising a sequence of nucleotides oramino acids, may comprise more nucleotides or amino acids than theactually cited ones, i.e., be embedded in a larger nucleic acid orprotein. A chimeric gene comprising a DNA region which is functionallyor structurally defined, may comprise additional DNA regions etc.

The invention is based on the one hand on the finding that eukaryoticcells, particularly plant cells, quite particularly Zea mays cellscontain simultaneously at least two functional major PARP proteinisoforms (classes) which differ in size and amino-acid sequence, yet areboth capable of binding DNA, particularly DNA with single strandedbreaks, and both have poly-ADP ribosylation activity. On the other hand,the inventors have realized that programmed cell death in eukaryotes,particularly in plants, can be modulated by altering the expressionlevel of the PARP genes or by altering the activity of the encodedproteins genetically, and that in order to achieve this goal, theexpression of both genes needs to be altered or in the alternative bothclasses of proteins need to be altered in their activity.

Thus, the invention relates to modulation—i.e. the enhancement or theinhibition—of programmed cell death or apoptosis in eukaryotic cells,preferably plant cells, by altering the level of expression of PARPgenes, or by altering the activity or apparent activity of PARP proteinsin that eukaryotic cell. Conveniently, the level of expression of PARPgenes or the activity of PARP proteins is controlled genetically byintroduction of PCD modulating chimeric genes altering the expression ofPARP genes and/or by introduction of PCD modulating chimeric genesaltering the apparent activity of the PARP proteins and/or by alterationof the endogenous PARP encoding genes.

As used herein, “enhanced PCD” with regard to specified cells, refers tothe death of those cells, provoked by the methods of the invention,whereby the killed cells were not destined to undergo PCD when comparedto similar cells of a normal plant not modified by the methods of theinvention, under similar conditions.

“Inhibited PCD” with regard to specified cells is to be understood asthe process whereby a larger fraction of those cells or groups of cells,which would normally (without the intervention by the methods of thisinvention) undergo programmed cell death under particular conditions,remain alive under those conditions.

The expression of the introduced PCD modulating chimeric genes or of themodified endogenous genes will thus influence the functional level ofPARP protein, and indirectly interfere with programmed cell death. Amoderate decrease in the functional level of PARP proteins leads to aninhibition of programmed cell death, particularly to prevention ofprogrammed cell death, while a severe decrease in the functional levelof the PARP proteins leads to induction of programmed cell death.

In accordance with the invention, it is preferred that in order toinhibit or prevent programmed cell death in a eukaryotic cell,particularly in a plant cell, the combined level of both PARP proteinsand/or their activity or apparent activity is decreased significantly,however avoiding that DNA repair (governed directly or indirectly byPARP) is inhibited in such a way that the cells wherein the function ofthe PARP proteins is inhibited cannot recover from DNA damage or cannotmaintain their genome integrity. Preferably, the level and/or activityof the PARP proteins in the target cells, should be decreased about 75%,preferably about 80%, particularly about 90% of the normal level and/oractivity in the target cells so that about 25%, preferably about 20%,particularly about 10% of the normal level and/or activity of PARP isretained in the target cells. It is further thought that the decrease inlevel and/or activity of the PARP proteins should not exceed 95%,preferably not exceed 90% of the normal activity and/or level in thetarget cells. Methods to determine the content of a specific proteinsuch as the PARP proteins are well known to the person skilled in theart and include, but are not limited to (histochemical) quantificationof such proteins using specific antibodies. Methods to quantify PARPactivity are also available in the art and include the above-mentionedTUNEL assay (in vivo) or the in vitro assay described Collinge andAlthaus (1994) for synthesis of poly (ADP-ribose) (see Examples).

Also in accordance with the invention, it is preferred that in order totrigger programmed cell death in a eukaryotic cell, particularly in aplant cell, the combined level of both PARP proteins and/or theiractivity or apparent activity is decreased substantially, preferablyreduced almost completely such that the DNA repair and maintenance ofthe genome integrity are no longer possible. Preferably, the combinedlevel and/or activity of the PARP proteins in the target cells, shouldbe decreased at least about 90%, preferably about 95%, more preferablyabout 99%, of the normal level and/or activity in the target cells,particularly the PARP activity should be inhibited completely. It isparticularly preferred that the functional levels of both classes ofPARP proteins separately are reduced to the mentioned levels.

For the purpose of the invention, PARP proteins are defined as proteinshaving poly (ADP-ribose) polymerase activity, preferably comprising theso-called “PARP signature”. The PARP signature is an amino acid sequencewhich is highly conserved between PARP proteins, defined by de Murciaand Menussier de Murcia (1994) as extending from amino acid at position858 to the amino acid at position 906 from the Mus musculus PARPprotein. This domain corresponds to the amino acid sequence fromposition 817 to 865 of the conventional PARP protein of Zea mays (ZAP1;SEQ ID No 2) or to the amino-acid sequence from position 827 to 875 ofthe conventional PARP protein of Zea mays (ZAP2; SEQ ID No 11) or to theamino acid sequence from position 500 to 547 of the non-conventionalPARP protein of Zea mays (SEQ ID No 4) or to the amino acid sequencefrom position 485 to 532 of the non-conventional PARP protein of.Arabidopsis thaliana (SEQ ID No 6). This amino sequence is highlyconserved between the different PARP proteins (having about 90% to 100%sequence identity). Particularly conserved is the lysine at position 891(corresponding to position 850 of SEQ ID No 2, position 861 of SEQ ID No11, position 532 of SEQ ID No 4, position 517 of SEQ ID No 6) of thePARP protein from Mus musculus, which is considered to be involved inthe catalytic activity of PARP proteins. Particularly the amino acids atposition 865, 866, 893, 898 and 899 of the PARP protein of Mus musculusor the corresponding positions for the other sequences are variable.PARP proteins may further comprise an N-terminal DNA binding domainand/or a nuclear localization signal (NLS).

Currently, two classes of PARP proteins have been described. The firstclass, as defined herein, comprises the so-called classical Zn-fingercontaining PARP proteins (ZAP). These proteins range in size from113-120 kDA and are further characterized by the presence of at leastone, preferably two Zn-finger domains located in the N-terminal domainof the protein, particularly located within the about 355 to about 375first amino acids of the protein. The Zn-fingers are defined as peptidesequences having the sequence CxxCx_(n)HxxC (whereby n may vary from 26to 30) capable of complexing a Zn atom. Examples of amino acid sequencesfor PARP proteins from the ZAP class include the sequences which can befound in the PIR protein database with accession number P18493 (Bostaurus), P26466 (Gallus gallus), P35875 (Drosophila melanogaster),P09874 (Homo sapiens), P11103 (Mus musculus), Q08824 (Oncorynchusmasou), P27008 (Rattus norvegicus), Q11208 (Sarcophaga peregrina),P31669 (Xenopus laevis) and the currently identified sequences of theZAP1 and ZAP2 protein from Zea mays (SEQ ID No 2/SEQ ID No 11).

The nucleotide sequence of the corresponding cDNAs can be found in theEMBL database under accession numbers D90073 (Bos taurus), X52690(Gallus gallus), D13806 (Drosophila melanogaster), M32721 (Homosapiens), X14206 (Mus musculus), D13809 (Oncorynchus masou), X65496(Rattus norvegicus), D16482 (Sarcophaga peregrina), D14667 (Xenopuslaevis) and in SEQ ID No 1 and 10 (Zea mays).

The second class as defined herein, comprises the so-callednon-classical PARP proteins (NAP). These proteins are smaller (72-73kDa) and are further characterized by the absence of a Zn-finger domainat the N-terminus of the protein, and by the presence of an N-terminaldomain comprising stretches of amino acids having similarity with DNAbinding proteins. Preferably, PARP protein of these class comprise atleast one amino acid sequence of about 30 to 32 amino acids whichcomprise the sequence R G x x x x G x K x x x x x R L (amino acids arerepresented in the standard one-letter code, whereby x stands for anyamino acid; SEQ ID No 7). Even more preferably these PARP proteinscomprise at least 1 amino acid sequence of about 32 amino acids havingthe sequence×L x V x x x R x x L x x R G L x x x V K x x L V x R L x x AI (SEQ ID No 8) (the so-called A1 domain) or at least 1 amino acidsequence of about 32 amino acids having the sequence G M x x x E L x x xA x x R G x x x x G x K K D x x R L x x (SEQ ID No 9)(the so-called A2domain) or both. Particularly, the A1 and A2 domain are capable offorming a helix-loop-helix structure. These PARP proteins may furthercomprise a basic “B” domain (K/R rich amino acid sequence of about 35 toabout 56 amino acids, involved in targeting the protein to the nucleus)and/or a an acid “C” domain (D/E rich amino acid sequence of about 36amino acids). Examples of protein sequences from the NAP class includethe APP protein from Arabidopsis thaliana (accessible from PIR proteindatabase under accession number Q11207; SEQ ID No 6) and the NAP proteinfrom Zea mays (SEQ ID No 4). The sequence of the corresponding cDNAs canbe found in the EMBL database under accession number Z48243 (SEQ ID No5) and in SEQ ID No 3. That the second class of PARP proteins are indeedfunctional PARP proteins, i.e. are capable of catalyzing DNA dependentpoly(ADP-ribose) polymerization has been demonstrated by the inventors(see Example 2).

The inventors have further demonstrated that eukaryotic cells,particularly plant cells express simultaneously genes encoding PARPproteins from both classes.

It is clear that for the purpose of the invention, other genes or cDNAsencoding PARP proteins from both classes as defined, or parts thereof,can be isolated from other eukaryotic species or varieties, particularlyfrom other plant species or varieties. These PARP genes or cDNAs can beisolated e.g. by Southern hybridization (either low-stringency orhigh-stringency hybridization depending on the relation between thespecies from which one intends to isolate the PARP gene and the speciesfrom which the probe was ultimately derived) using as probes DNAfragments with the nucleotide sequence of the above mentioned PARP genesor cDNAs, or parts thereof, preferably parts which are conserved such asa gene fragment comprising the nucleotide sequence encoding the PARPsignature mentioned supra. The nucleotide sequences corresponding to thePARP signature from the PARP proteins encoded by plant genes are thenucleotide sequence of SEQ ID No 1 from nucleotide 2558 to 2704 or thenucleotide sequence of SEQ ID No 3 from nucleotide 1595 to 1747 or thenucleotide sequence of SEQ ID No 5 from nucleotide 1575 to 1724. If adiscrimination is to be made between the classes of PARP genes, parts ofthe PARP genes which are specific for the class, such as the N-terminaldomains preceding the catalytic domain or parts thereof, shouldpreferably be used.

Alternatively, the genes or cDNAs encoding PARP proteins or partsthereof, can also be isolated by PCR-amplification using appropriateprimers such as the degenerated primers with the nucleotide sequencecorresponding to the sequences indicated in SEQ ID No 13, SEQ ID No 14,or primers with the nucleotide sequence corresponding to the sequencesindicated in SEQ ID No 15 to 20. However, it is clear that the personskilled in the art can design alternative oligonucleotides for use inPCR or can use oligonucleotides comprising a nucleotide sequence of atleast 20, preferably at least about 30, particularly at least about 50,consecutive nucleotides of any of the PARP genes to isolate the genes orpart there of by PCR amplification.

It is clear that a combination of these techniques, or other techniques(including e.g. RACE-PCR), available to the skilled artisan to isolategenes or cDNAs on the basis of partial fragments and their nucleotidesequence, e.g. obtained by PCR amplification, can be used to isolatePARP genes, or parts thereof, suitable for use in the methods of theinvention.

Moreover, PARP genes, encoding PARP proteins wherein some of the aminoacids have been exchanged for other, chemically similar, amino acids(so-called conservative substitutions), or synthetic PARP genes (whichencode similar proteins as natural PARP genes but with a differentnucleotide sequence, based on the degeneracy of the genetic code) andparts thereof are also suited for the methods of the invention.

In one aspect of the invention, PCD in eukaryotic cells, particularly inplant cells, is inhibited by a moderate decrease in the functional levelof PARP in those eukaryotic cells.

In one embodiment of this first aspect of the invention, the functionallevel of PARP in eukaryotic cells, particularly in plant cells isreduced by introduction of at least one PCD modulating chimeric gene inthose cells, comprising a promoter capable of directing transcription inthese cells, preferably a plant-expressible promoter, and a functional3′ transcription termination and polyadenylation region, operably linkedto a DNA region which when transcribed yields a biologically active RNAmolecule which is capable of decreasing the functional level of theendogenous PARP activity encoded by both classes of PARP genes.

In a preferred embodiment, at least two such PCD modulating chimericgenes are introduced in the cells, whereby the biologically active RNAencoded by the first PCD modulating chimeric gene decreases thefunctional level of the endogenous PARP activity encoded by the genes ofthe NAP class, and whereby the biologically active RNA encoded by thesecond PCD modulating chimeric gene decreases the functional level ofthe endogenous PARP activity encoded by the genes of the ZAP class, sothat the combined PARP activity is moderately decreased.

In a particularly preferred embodiment, the PCD modulating chimericgenes decrease the functional level of the endogenous PARP activity byreducing the level of expression of the endogenous PARP genes. To thisend, the transcribed DNA region encodes a biologically active RNA whichdecreases the mRNAs encoding NAP and ZAP class PARP proteins, that isavailable for translation. This can be achieved through techniques suchas antisense RNA, co-suppression or ribozyme action.

As used herein, “co-suppression” refers to the process oftranscriptional and/or post-transcriptional suppression of RNAaccumulation in a sequence specific manner, resulting in the suppressionof expression of homologous endogenous genes or transgenes.

Suppressing the expression of the endogenous PARP genes can thus beachieved by introduction of a transgene comprising a strong promoteroperably linked to a DNA region whereby the resulting transcribed RNA isa sense RNA or an antisense RNA comprising a nucleotide sequence whichhas at least 75%, preferably at least 80%, particularly at least 85%,more particularly at least 90%, especially at least 95% sequenceidentity with or is identical to the coding or transcribed DNA sequence(sense) or to the complement (antisense) of part of the PARP gene whoseexpression is to be suppressed. Preferably, the transcribed DNA regiondoes not code for a functional protein. Particularly, the transcribedregion does not code for a protein. Further, the nucleotide sequence ofthe sense or antisense region should preferably be at least about 100nucleotides in length, more preferably at least about 250 nucleotides,particularly at least about 500 nucleotides but may extend to the fulllength of the coding region of the gene whose expression is to bereduced.

For the purpose of this invention the “sequence identity” of two relatednucleotide or amino acid sequences, expressed as a percentage, refers tothe number of positions in the two optimally aligned sequences whichhave identical residues (×100) divided by the number of positionscompared. A gap, i.e. a position in an alignment where a residue ispresent in one sequence but not in the other is regarded as a positionwith non-identical residues. The alignment of the two sequences isperformed by the Wilbur and Lipmann algorithm (Wilbur and Lipmann, 1983)using a window-size of 20 nucleotides or amino acids, a word length of 2amino acids, and a gap penalty of 4. Computer-assisted analysis andinter relation of sequence data, including sequence alignment asdescribed above, can be conveniently performed using commerciallyavailable software packages such as the programs of the Intelligenetics™Suite (Intelligenetics Inc., CA).

It will be clear to a skilled artisan that one or more sense orantisense PCD modulating chimeric genes can be used to achieve the goalsof the first aspect of the invention. When one sense or antisense PCDmodulating chimeric gene is used, this gene must be capable ofsimultaneously reducing the expression of PARP genes of both classes.This can e.g. be achieved by choosing the transcribed region of thechimeric gene in such a way that expression of both classes of genes canbe regulated by one sense or antisense RNA, i.e. by choosing targetregions corresponding to the highest homology DNA region of the PARPgenes of both classes and incorporating a sense or antisense transcribedDNA region corresponding to both target regions, conform to theconditions described above for sense and antisense RNA. Alternatively,different sense or antisense RNA regions, each specific for regulatingthe expression of one class of PARP genes, can be combined into one RNAmolecule, encoded by one transcribed region of one PCD modulatingchimeric gene. Obviously, the different sense or antisense RNA regionsspecific for regulating the expression of one class of PARP genes can beintroduced as separate PCD modulating chimeric genes.

Preferred sense and antisense encoding transcribed regions comprise anucleotide sequence corresponding (with sequence identity constraints asindicated above) to a sequence of at least about 100 consecutivenucleotides selected from the N-terminal domains of the PARP genes,preferably corresponding to a sequence of at least about 100 consecutivenucleotides selected from the sequence of SEQ ID No 1 from nucleotideposition 113 to 1189, the sequence of SEQ ID No 3 from nucleotideposition 107 to 583, the sequence of SEQ ID No 5 from nucleotideposition 131 to 542 or the sequence of SEQ ID No 10 from nucleotideposition 81 to 1180. However, it is clear that sense or antisenseencoding transcribed regions can be used comprising a sequencecorresponding to the complete sequence of the N-terminal domain of thePARP genes, or even to complete sequence of the PARP genes, particularlythe protein-encoding region thereof. Further preferred are sense andantisense encoding transcribed regions which comprise a nucleotidesequence corresponding (with sequence identity constraints as indicatedabove) to a sequence of at least about 100 consecutive nucleotidesselected from the C-terminal catalytic domains of the PARP genes,preferably a sequence of at least 100 nucleotides encompassing thePARP-signature encoding nucleotide sequences, particularly thePARP-signature encoding nucleotide sequences indicated supra. Again, itis clear that sense or aritisense encoding transcribed regions can beused comprising a sequence corresponding to the complete sequence of theC-terminal domain of the PARP genes.

In another particularly preferred embodiment, the PCD modulatingchimeric genes decrease the functional level of the endogenous PARPactivity by reducing the level of apparent activity of the endogenousPARPs of both classes. To this end, the transcribed DNA region encodes abiologically active RNA which is translated into a protein or inhibitingNAP or ZAP class PARP proteins or both, such as inactivating antibodiesor dominant negative PARP mutants.

“Inactivating antibodies of PARP proteins” are antibodies or partsthereof which specifically bind at least to some epitopes of PARPproteins, such as the epitope covering part of the ZN finger 11 fromposition 111-118 in ZAP1 or a corresponding peptide in ZAP2, and whichinhibit the activity of the target protein.

“Dominant negative PARP mutants” as used herein, are proteins orpeptides comprising at least part of a PARP protein (or a variantthereof), preferably a PARP protein endogenous to the eukaryotic targethost cell, which have no PARP activity, and which have an inhibitoryeffect on the activity of the endogenous PARP proteins when expressed inthat host cell. Preferred dominant negative PARP mutants are proteinscomprising or consisting of a functional DNA binding domain (or avariant thereof) without a catalytic domain (such as the N-terminalZn-finger containing domain of about 355 to about 375 amino acids of aPARP: of the ZAP class, particularly a DNA binding protein domaincomprising the amino acid sequence of SEQ ID No 2 from amino acid 1 to370 or a DNA binding protein domain comprising the amino acid sequenceof SEQ ID No 11 from amino acid 1 to 98, or a DNA binding protein domaincomprising the amino acid sequence of SEQ ID No 2 from amino acid 1 to370 wherein the amino acid sequence from amino acid 1 to 88 is replacedby the amino acid sequence of SEQ ID No 11 from amino acid at position 1to the amino acid at position 98, or such as the N-terminal DNA bindingprotein domain of about 135 to 160 amino acids of a PARP of the NAPclass, particularly a DNA binding protein domain comprising the aminoacid sequence of SEQ ID No 4 from amino acid 1 to 159 or a DNA bindingprotein domain comprising the amino acid sequence of SEQ ID No 6 fromamino acid 1 to 138) or without a functional catalytic domain (such asinactive PARP mutants, mutated in the so-called PARP signature,particularly mutated at the conserved lysine of position 850 of SEQ IDNo 2, position 532 of SEQ ID No 4, position 517 of SEQ ID No 6).Preferably, dominant negative PARP mutants should retain their DNAbinding activity. Dominant negative PARP mutants can be fused to acarrier protein, such as a β-glucuronidase (SEQ ID No 12).

Again, one or more PCD modulating genes encoding one or more dominantnegative PARP mutants can be used to achieve the goals of the firstaspect of the invention. When one PCD modulating chimeric gene is used,this gene must be capable of simultaneously reducing the expression ofPARP genes of both classes.

In another embodiment of the first aspect of the invention, thefunctional level of PARP in eukaryotic cells, particularly in plantcells is reduced by modification of the nucleotide sequence of theendogenous PARP genes in those cells so that the encoded mutant PARPproteins retain about 10% of their activity. Methods to achieve such amodification of endogenous PARP genes include homologous recombinationto exchange the endogenous PARP genes for mutant PARP genes e.g. by themethods described in U.S. Pat. No. 5,527,695. In a preferred embodimentsuch site-directed modification of the nucleotide sequence of theendogenous PARP genes is achieved by introduction of chimeric DNA/RNAoligonucleotides as described in WO 96/22364 or U.S. Pat. No. 5,565,350.

In another aspect of the invention, programmed death of eukaryoticcells, preferably selected cells, particularly selected plant cells isenhanced by a severe decrease in the functional level of PARP,preferably reduced-almost completely, such that the DNA repair andmaintenance of the genome integrity are no longer possible.

In one embodiment of this aspect of the invention, the functional levelof PARP in eukaryotic cells, particularly in plant cells is reducedseverely, particularly abolished almost completely, by introduction ofat least one PCD modulating chimeric gene in those cells, comprising apromoter capable of directing transcription in these cells, preferably aplant-expressible promoter, and a functional 3′ transcriptiontermination and polyadenylation region, operably linked to a DNA regionwhich when transcribed yields a biologically active RNA molecule whichis capable of decreasing the functional level of the endogenous PARPactivity encoded by both classes of PARP genes.

In a preferred embodiment of the second aspect of the invention, atleast two such PCD modulating chimeric genes are introduced in thecells, whereby the biologically active RNA encoded by the first PCDmodulating chimeric gene decreases the functional level of theendogenous PARP activity encoded by the genes of the NAP class, andwhereby the biologically active RNA encoded by the second PCD modulatingchimeric gene decreases the functional level of the endogenous PARPactivity encoded by the genes of the ZAP class, so that the combinedPARP activity is severely decreased, particularly almost completelyeliminated.

As mentioned for the first aspect of this invention, the transcribedregions of the PCD modulating chimeric genes encode biologically activeRNA, which can interfere with the expression of the endogenous PARPgenes (e.g. through antisense action, co-suppression or ribozyme action)or the biologically active RNA can be further translated into a peptideor protein, capable of inhibiting the PARP proteins of the NAP and ZAPclass, such as inactivating antibodies or dominant negative PARPmutants.

In a particularly preferred embodiment of the second aspect of theinvention, the transcribed region of the PCD modulating chimeric genes(PCD enhancing chimeric genes) codes for a biologically active RNA whichcomprises at least one RNA region (preferably of at least about 100nucleotides in length) classifying according to the above mentionedcriteria as a sense RNA for at least one of the endogenous PARP genes,and at least one other RNA region (preferably of at least about 100nucleotides in length), classifying according to the above mentionedcriteria as an antisense RNA for at least one of the endogenous PARPgenes, whereby the antisense and sense RNA region are capable ofcombining into a double stranded RNA region (preferably over a distanceof at least about 100 nucleotides). In an especially preferredembodiment, two such PCD modulating genes, one targeted to reduce thefunctional level of a PARP protein of the NAP class, and the othertargeted to reduce the functional level of a PARP protein of the ZAPclass are introduced into an eukaryotic cell or organism, preferably aplant cell or plant.

It is clear that the different embodiments for the transcribed DNAregions of the chimeric PCD modulating genes of the invention can beused in various combinations to arrive at the goals of the invention.E.g. a first chimeric PCD modulating gene may encode a sense RNAdesigned to reduce the expression of an endogenous PARP gene of the ZAPclass, while the second chimeric PCD modulating gene may encode adominant negative PARP mutant designed to reduce the expression of anendogenous PARP gene of the NAP class.

Whether the introduction of PCD modulating chimeric genes intoeukaryotic cells will ultimately result in a moderately reduced or aseverally reduced functional level of combined PARP in those cells—i.e.in inhibited PCD or enhanced PCD—will usually be determined by theexpression level (either on transcriptional level or combinedtranscriptional/translational level) of those PCD modulating genes. Amajor contributing factor to the expression level of the PCD modulatinggene is the choice of the promoter region, although other factors (suchas, but not limited to, the choice of the 3′ end, the presence ofintrons, codon usage of the transcribed region, mRNA stability, presenceof consensus sequence around translation initiation site, choice of 5′and 3′ untranslated RNA regions, presence of PEST sequences, theinfluence of chromatin structure surrounding the insertion site of astabile integrated PCD modulating gene, copy number of the introducedPCD modulating genes, etc.) or combinations thereof will also contributeto the ultimate expression level of the PCD modulating gene. In general,it can be assumed that moderate reduction of functional levels ofcombined PARP can be achieved by PCD modulating genes comprising arelatively weak promoter, while severe reduction of functional levels ofcombined PARP can be achieved by PCD modulating genes comprising arelatively strong promoter. However, the expression level of a PCDmodulating gene comprising a specific promoter and eventually its effecton PCD, can vary as a function of the other contributing factors, asalready mentioned.

For the purpose of particular embodiments of the invention, the PCDmodulating chimeric genes may comprise a constitutive promoter, or apromoter which is expressed in all or the majority of the cell typesthroughout the organism, particularly throughout the plant, such as thepromoter regions derived from the T-DNA genes, particularly the opinesynthase genes of Agrobacterium Ti- or Ri-plasmids (e.g. nos, ocspromoters), or the promoter regions of viral genes (such as CaMV35Spromoters, or variants thereof).

It may be further be advantageous to control the expression of the PCDmodulating gene at will or in response to environmental cues, e.g. byinclusion of an inducible promoter which can be activated by an externalstimuli, such as, but not limited to application of chemical compounds(e.g. safeners, herbicides, glucocorticoids), light conditions, exposureto abiotic stress (e.g. wounding, heavy metals, extreme temperatures,salinity or drought) or biotic stress (e.g. pathogen or pest infectionincluding infection by fungi, viruses, bacteria, insects, nematodes,mycoplasms and mycoplasma like organisms etc.). Examples ofplant-expressible inducible promoters suitable for the invention are:nematode inducible promoters (such as disclosed in WO 92/21757), fungusinducible promoters (WO 93/19188, WO 96/28561), promoters inducibleafter application of glucocorticoids such as dexamethasone ( ), orpromoters repressed or activated after application of tetracyclin (Gatzet al. 1988; Weimann et al 1994)

In several embodiments of the invention, particularly for the secondaspect of the invention (i.e. enhanced PCD), it may be convenient orrequired to restrict the effect on programmed cell death to a particularsubset of the cells of the organism, particularly of the plant, hencethe PCD modulating genes may include tissue-specific or celltype-specific promoters. Examples of suitable plant-expressiblepromoters selectively expressed in particular tissues or cell types arewell known in the art and include but are not limited to seed-specificpromoters (e.g. WO89/03887), organ-primordia specific promoters (An etal., 1996), stem-specific promoters (Keller et al., 1988), leaf specificpromoters (Hudspeth et al., 1989), mesophyl-specific promoters (such asthe light-inducible Rubisco promoters), root-specific promoters (Kelleret al., 1989), tuber-specific promoters (Keil et al., 1989), vasculartissue specific promoters (Peleman et al., 1989), meristem specificpromoters (such as the promoter of the SHOOTMERISTEMLESS (STM) gene,Long et al., 1996), primordia specific promoter (such as the promoter ofthe Antirrhinum CycD3a gene, Doonan et al., 1998), anther specificpromoters (WO 89/10396, WO9213956, WO9213957) stigma-specific promoters(WO 91/02068), dehiscence-zone specific promoters (WO 97/13865),seed-specific promoters (WO 89/03887) etc.

Preferably the chimeric PCD modulating genes of the invention areaccompanied by a marker gene, preferably a chimeric marker genecomprising a marker DNA that is operably linked at its 5′ end to aplant-expressible promoter, preferably a constitutive promoter, such asthe CaMV ³⁵S promoter, or a light inducible promoter such as thepromoter of the gene encoding the small subunit of Rubisco; and operablylinked at its 3′ end to suitable plant transcription 3′ end formationand polyadenylation signals. It is expected that the choice of themarker DNA is not critical, and any suitable marker DNA can be used. Forexample, a marker DNA can encode a protein that provides adistinguishable “color” to the transformed plant cell, such as the A1gene (Meyer et al., 1987) or Green Fluorescent Protein (Sheen et al.,1995), can provide herbicide resistance to the transformed plant cell,such as the bar gene, encoding resistance to phosphinothricin (EP0,242,246), or can provided antibiotic resistance to the transformedcells, such as the aac(6′) gene, encoding resistance to gentamycin(WO94/01560).

Methods to introduce PCD modulating chimeric genes into eukaryoticcells, particularly methods to transform plant cells are well known inthe art, and are believed not to be critical for the methods of theinvention. Transformation results in either transient or stablytransformed cells (whereby the PCD modulating chimeric genes are stablyinserted in the genome of the cell, particularly in the nuclear genomeof the cell).

It is clear that the methods and means described in this invention toalter the programmed cell death in eukaryotic cells and organisms,particularly in plant cells and plants, has several importantapplication possibilities. Inhibition of PCD by the methods and means ofthe invention, can be used to relieve the stress imposed upon the cells,particularly the plant cells, during transformation and thus to increasetransformation efficiency, as described in WO 97/06267. Inhibition ofPCD can also be used to improve cell culture of eukaryotic cells,particularly of plant cells. Triggering of PCD in particular cell typesusing the means and methods of the invention, can be used for methodswhich call upon the use of a cytotoxin. Since PCD is the “natural” wayfor cells to die, the use of PCD enhancing chimeric genes of theinvention constitutes an improvement over the use of other cytotoxicgenes such as RNAse or diptheria toxin genes which lead to cell lysis.Moreover, low-level expression of PCD enhancing genes in cells differentthan the targeted cells, will lead to a moderate reduction instead of asevere reduction of PARP activity in those cells, thus actuallyinhibiting PCD in non-target cells.

For plants, preferred applications of PCD enhancing chimeric genesinclude, but are not limited to:

-   1. the generation of plants protected against fungus infection,    whereby the PCD enhancing chimeric gene or genes comprise a    fungus-responsive promoter as described in WO 93/19188 or WO    96/28561.-   2. the generation of nematode resistant plants, whereby the PCD    enhancing chimeric gene or genes comprise a nematode inducible    promoters such as disclosed in WO 92/21757-   3. the generation of male or female sterile plants, whereby the PCD    enhancing chimeric gene or genes comprise anther-specific promoters    (such as disclosed in WO 89/10396, WO9213956, WO9213957) or    stigma-specific promoters (such as disclosed in WO 91/02068)-   4. the generation of plants with improved seed shatter    characteristics whereby the PCD enhancing chimeric gene or genes    comprise dehiscence zone-specific promoters (such as disclosed in WO    97/13865).

Although it is clear that the invention can be applied essentially toall plant species and varieties, the invention will be especially suitedto alter programmed cell death in plants with a commercial value.Particularly preferred plants to which the invention can be applied arecorn, oil seed rape, linseed, wheat, grasses, alfalfa, legumes, abrassica vegetable, tomato, lettuce, cotton, rice, barley, potato,tobacco, sugar beet, sunflower, and ornamental plants such as carnation,chrysanthemum, roses, tulips and the like.

The obtained transformed plant can be used in a conventional breedingscheme to produce more transformed plants with the same characteristicsor to introduce the chimeric cell-division controlling gene of theinvention in other varieties of the same or related plant species. Seedsobtained from the transformed plants contain the PCD modulating gene ofthe invention as a stable genomic insert.

The following non-limiting Examples describe the construction ofchimeric apoptosis controlling genes and the use of such genes for themodulation of the programmed cell death in eukaryotic cells andorganisms. Unless stated otherwise in the Examples, all recombinant DNAtechniques are carried out according to standard protocols as describedin Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2of Ausubel et al. (1994) Current Protocols in Molecular Biology, CurrentProtocols, USA. Standard materials and methods for plant molecular workare described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy,jointly published by BIOS Scientific Publications Ltd (UK) and BlackwellScientific Publications, UK.

Throughout the description and Examples, reference is made to thefollowing sequences:

-   SEQ ID No 1: DNA sequence of the ZAP gene of Zea mays (zap1)-   SEQ ID No 2: protein sequence of the ZAP protein of Zea mays (ZAP1)-   SEQ ID No 3: DNA sequence of the NAP gene of Zea mays (nap)-   SEQ ID No 4: protein sequence of the NAP protein of Zea mays (NAP)-   SEQ ID No 5: DNA sequence of the NAP gene of Arabidopsis thaliana    (app)-   SEQ ID No 6: protein sequence of the NAP protein of Arabidopsis    thaliana (APP)-   SEQ ID No 7: consensus sequence for the A domain of non-conventional    PARP proteins-   SEQ ID No 8: consensus sequence for the A1 domain of    non-conventional PARP proteins-   SEQ ID No 9: consensus sequence for the A2 domain of    non-conventional PARP proteins-   SEQ ID No 10: DNA sequence of the second ZAP gene of Zea mays (Zap2)-   SEQ ID No 11: protein sequence of the ZAP protein of Zea mays (ZAP2)-   SEQ ID No 12: amino acid sequence of a fusion protein between the    DNA binding domain of APP and the GUS protein-   SEQ ID No 13: degenerated PCR primer-   SEQ ID No 14: degenerated PCR primer-   SEQ ID No 15: PCR primer-   SEQ ID No 16: PCR primer-   SEQ ID No 17: PCR primer-   SEQ ID No 18: PCR primer-   SEQ ID No 19: PCR primer-   SEQ ID No 20: PCR primer-   SEQ ID No 21: app promoter-gus translational fusion    Sequence Listing Free Text

The following free text has been used in the Sequence Listing part ofthis application

-   <223> Description of Artificial Sequence: A domain of    non-conventional PARP proteins-   <223> Description of Artificial Sequence: A1 domain on non    conventional PARP protein-   <223> Description of Artificial Sequence: A2 domain of    non-conventional PARP protein-   <223> Description of Artificial Sequence: fusion protein between APP    N-terminal domain and GUS protein-   <223> Description of Artificial Sequence: degenerated PCR primer-   <223> Description of Artificial Sequence: oligonucleotide for use as    PCR primer-   <223> Description of Artificial Sequence: APP promoter fusion with    beta-glucuronidase gene-   <223> translation initiation codon

EXAMPLES Experimental Procedures

Yeast and Bacterial Strains

Saccharomyces cerevisiae strain DY (MATa his3 canl-10 ade2 leu2 trp1ura3::(3xSV40 AP1-lacZ) (Kuge and Jones, 1994) was used for theexpression of the APP protein. Yeast transformation was carried outaccording to Dohmen et al (1991). Strains were grown on a minimal SDCmedium (0.67% yeast nitrogen base, 0.37% casamino acids, 2% glucose, 50mgl⁻¹ of adenine and 40 mgl⁻¹ of tryptophan). For the induction of theAPP expression, glucose in SDC was substituted with 2% galactose.

Escherichia coli strain XL-I (Stratagene, La Jolla, Calif.) was used forthe plasmid manipulations and library screenings, which were carried outaccording to standard procedures (Ausubel et al., 1987; Sambrook et al.,1989). E. coli BL21 (Studier and Moffat, 1986) was used for the APPprotein expression and Agrobacterium tumefaciens C58C1 Rif^(R)(pGV2260)(Deblaere et al., 1985) for the stable transformation of plants.

Poly(ADP-ribose)Polymerase Activity Assay

Enzymatic activity of the APP was assayed in total protein extracts ofyeast strains prepared as follows. DY(pV8SPA) or DY(pYeDP1/8-2) weregrown in 50 ml of SDC medium overnight at 30° C. on a gyratory shaker at150 rpm. Yeast cells were harvested by centrifugation at 1,000×g, washedthree times with 150 ml of 0.1 M potassium phosphate buffer (pH 6.5),and resuspended in 5 ml of sorbitol buffer (1.2 M sorbitol, 0.12 MK₂HPO₄, 0.033 M citric acid, pH 5.9). Lyticase (Boehringer, Mannheim,Germany) was added to the cell suspension to a final concentration of 30U ml⁻¹ and cells were incubated at 30° C. for 1 h. Yeast spheroplastswere then washed three times with sorbitol buffer and resuspended in 2ml of ice-cold lysis buffer (100 mM Tris-HCl, pH 7.5, 400 mM NaCl, 1 mMEDTA, 10% glycerol, 1 mM DTT). After sonication, the lysate wascentrifuged at 20,000×g for 20 min at 4° C. and the supernatant wasdesalted on a Econo-Pack™ 10 DG column (Bio-Rad, Richmond, Calif.)equilibrated with reaction buffer (100 mM Tris-HCl, pH 8.0, 10 mM MgCl₂,1 mM DTT). To reduce proteolytic degradation of proteins, the lysis andreaction buffers were supplemented with a protease inhibitor cocktail(Boehringer), one tablet per 50 ml. Nucleic acids were removed from thetotal extracts by adding NaCl and protamine sulfate to a finalconcentration of 600 mM and 10 mg ml¹, respectively. After incubation atroom temperature for 10 min, the precipitate was removed bycentrifugation at 20,000×g for 15 min at 4° C. The buffer of thesupernatant was exchanged for the reaction buffer by gel filtration onan Econo-Pack™ 10 DG column.

The assay for the synthesis of poly(ADP-ribose) was adapted fromCollinge and Althaus (1994). Approximately 500 μg of total yeast proteinwere incubated in a reaction buffer supplemented with 30 μCi of ³²P-NAD⁺(500 Ci mmol⁻¹), unlabeled NAD⁺ to a final concentration of 60 μM, and10 μg ml⁻¹ sonicated salmon sperm DNA. After incubation for 40 min atroom temperature, 500 μl of the stop buffer (200 mM Tris-HCl, pH 7.6,0.1 M NaCl, 5 mM EDTA, 1% Na⁺-N-lauroyl-sarcosine, and 20 μg ml⁻¹proteinase K) were added and reactions incubated at 37° C. overnight.After phenol and phenol/chloroform extractions, polymers wereprecipitated with 2.5 volumes of ethanol with 0.1 M NaAc (pH 5.2). Thepellet was washed with 70% ethanol, dried, and dissolved in 70%formamide, 10 mM EDTA, 0.01% bromophenol blue, and 0.01% xylene cyanol.Samples were heated at 80° C. for 10 min and then loaded onto a 12%polyacrylamide/6 M urea sequencing gel. Gels were dried on 3 MM paper(Whatman International, Maidstone, UK) and exposed either to KodakX-Omat X-ray film (Eastman Kodak, Richmond, N.Y.) or scanned using aPhosphorlmager™445SI (Molecular Dynamics, Sunnyvale, Calif.).

Immunological Techniques

A truncated app cDNA encoding an APP polypeptide from amino acids Met³¹⁰to His⁶³⁷ was expressed as a translation fusion with six histidineresidues at the N terminus after induction of a 500-ml culture of the E.coli BL21(pETΔNdeSPA) with 1 mM isopropyl-β-D-thiogalactopyranoside. TheAPP polypeptide was purified to near homogeneity by affinitychromatography under denaturing conditions (in the presence of 6 Mguanidinium hydrochloride) on a Ni²⁺-NTA-agarose column, according tothe manufacturers protocol (Qiagen, Chatsworth, Calif.). After dialysisagainst PBS, a mixture of the soluble and insoluble APP polypeptides wasused to immunize two New Zealand White rabbits following a standardimmunization protocol (Harlow and Lane, 1988). For the Western blotanalysis, proteins were resolved by denaturing SDS-PAGE (Sambrook etal., 1989; Harlow and Lane, 1988) and transferred onto nitrocellulosemembranes (Hybond-C; Amersham), using a Semi-Dry Blotter II (Kem-En-Tec,Copenhagen, Denmark).

In situ antigen localization in yeast cells was carried out as described(Harlow and Lane, 1988). For the localization of the APP protein inyeast spheroplasts, anti-APP serum was diluted 1:3,000 to 1:5,000 inTris-buffered saline-BSA buffer. 10H monoclonal antibody, whichspecifically recognizes poly(ADP-ribose) polymer (Ikajima et al., 1990)was used in a 1:100 dilution in PBS buffer. The mouse antibody weredetected with the sheep anti-mouse IgG F(ab′)₂ fragment conjugated tofluorescein isothiocyanate (FITC) (Sigma) at a dilution of 1:200. RabbitIgG was detected with CY-3 conjugated sheep anti-rabbit IgG sheep F(ab)₂fragment (Sigma), at a dilution of 1:200. For the visualization of DNA,slides were incubated for 1 min in PBS with 10 μg ml⁻¹ of4′,6-diamidino-2-phenylindole (DAPI; Sigma). Fluorescence imaging wasperformed on an Axioskop epifluorescence microscope (Zeiss, Jena,Germany). For observation of FITC and CY-3 fluorochromes, 23 and 15filter cubes were used, respectively. Cells were photographed with FujiColor-100 super plus film.

Plant Material and Histochemical Analysis

Nicotiana tabacum SR1 (Maliga et al., 1975) was used for the generationof stable transformants following the procedure of leaf disccocultivation (De Block et al., 1987) with A. tumefaciens C58C1 Rif^(R)(pGV2260; PGCNSPAGUS). N. tabacum SR1 line transformed with authenticGUS under the control of the 35S CaMV was used as a control. Arabidopsisthaliana ecotype Columbia was used for the transformation of theapp-promoter-GUS fusion following the in situ infiltration procedure.

For in situ histochemical staining of the GUS activity, plant sampleswere fixed in ice-cold 90% acetone for 30 min, washed in 0.1 M K₂HPO₄(pH 7.8), and then incubated in staining buffer (0.1 M K₂HPO₄, pH 7.8, 2mM X-Gluc, 20 mM Fe³⁺-EDTA) at 37° C. Stained plant tissues were storedin 70% ethanol at 4° C. When necessary, browning of tissues due tophenolic oxidation was reduced by incubation with lactophenol (Beeckmanand Engler, 1994). The GUS staining was examined under a Jenalumar lightmicroscope (Zeiss). Plant tissues were photographed with Fuji Color-100super plus film.

Miscellaneous Methods

The plasmid construction steps were routinely verified by DNA sequencingcarried out according to protocols provided by USB Biochemicals(Cleveland, Ohio). ³²P-labeled DNA probes for nucleic acid hybridizationwere synthesized by the Ready-Prime DNA labelling kit (Amersham). ForDNA and RNA hybridization experiments, the buffer system of Church andGilbert (1984) was used (0.25 M sodium phosphate, pH 7.2, 7% SDS, 1%BSA, 1 mM EDTA). For Western blot analysis, yeast total proteins wereextracted with phenol essentially as described for plant tissues(Hurkman and Tanaka, 1986). For Northern blot analysis, total yeast RNAwas extracted with hot phenol as described (Ausubel et al., 1987). RNAwas resolved on 1.5% agarose gels after denaturation with glyoxal(Sambrook et al., 1989). Hybond-N nylon filters (Amersham) were used forthe nucleic acid blotting.

Example 1 Isolation of Genes Encoding PARP Homologues from Zea Mays

With the purpose of isolating maize cDNA encoding PARP homologue(s) twoapproaches were followed. First, a maize cDNA library was screened underlow-stringency DNA—DNA hybridization conditions using a DNA probeprepared from the Arabidopsis app cDNA. Secondly, PCR amplification ofpart of the maize PARP was performed, using the first-strand cDNA as atemplate and two degenerate primers, designed on the basis of thesequence of the “PARP signature” the most conserved amino acid sequencebetween all known PARP proteins.

A λZAP (Stratagene) cDNA library from leaves of maize (Zea mays L.),inbred line B734. Plaques (500,000) were screened according to standardprocedures (Sambrook et al., 1989). After screening with the Arabidopsisapp probe, one non-full-length cDNA of 1.4 kbp was purified. After theinitial cDNA library screening with the app probe and a subsequent 5′rapid amplification of cDNA ends (RACE) PCR analysis, the nap gene, amaize homologue of the Arabidopsis app, was identified. For the 5′RACEPCR, the template was prepared with the Marathon kit (Clontech, PaloAlto, Calif.) and 0.5 μg of maize poly(A)⁺ RNA isolated from innersheath, outer sheath, and leaves of 1-week-old maize seedlings. Thegene-specific, nested primers for PCR amplification were5′-GGGACCATGTAGTTTATCTTGACCT-3′ (SEQ ID No 15) and5′-GACCTCGTACCCCAACTCTTCCCCAT-3′ (SEQ ID No 16) for nap primers. Theamplified PCR products were subcloned and sequenced. A fragment of 800bp was amplified with nap specific primers which allowed to reconstructthe 2295-bp-long sequence of nap cDNA (SEQ ID No 3).

The NAP protein was 653 amino acids long (molecular mass ˜73 kDa; SEQ IDNo 4) and highly similar (61% sequence identity and 69% similarity) tothe APP. Most importantly, NAP had an organization of the N-terminuscongruent to APP (FIG. 1A), suggesting a rather strict selectionpressure on the structure of APP-like proteins in plants. The nap genewas unique in the maize genome (FIG. 2A) and encoded a transcript of 2.4kb (FIG. 2C).

Using degenerate primers based on very highly conserved regions in the“PARP signature” and first-strand cDNA from Zea mays as a template, a310-bp fragment was amplified. For the PCR with degenerate primers5′-CCGAATTCGGNTAYATGTTYGGNAA-3′ (SEQ ID No 13) and5′-CCGAATTCACNATRTAYTCRTTRTA-3′ (SEQ. ID No 14) with Y═C/T; R=A/G;N=A/G/C/T), the first strand cDNA was used as a template and wassynthesized using 5 μg of poly(A)⁺ RNA from young maize leaves and MuMLVreverse transcriptase. PCR amplifications were performed with Taq DNApolymerase in 100 μl volume using the following conditions: 1 min at 95°C., 2 min at 45° C., 3 min at 72° C., followed by 38 cycles of 1 min at95° C., 2 min at 45° C., 3 min at 72° C., with a final incubation for 10min at 72° C.

The sequence of the 310 bp fragment showed 55% sequence identity and 64%sequence similarity with human PARP over the same region, but was,however, different from the sequence of the nap cDNA. Three zap cDNAswere identified after screening with the 310-bp fragment, which wasobtained by PCR with degenerate primers. These three purified cDNA wereall derived from the same transcript because they had identical 3′non-coding regions; the longest clone (#9) was sequenced on both strands(SEQ ID No 1). This cDNA encoded a PARP-homologous polypeptide of 689amino acids (SEQ ID No 2; molecular mass ˜109 kDa), which we designatedas ZAP1 (FIG. 1B). The first Zn-finger of ZAP1 was probablynonfunctional because it had the sequence CKSCxxxHASV, which included nothird cysteine residue.

5′RACE PCR analysis of zap transcripts from the maize line LG2080 (thescreened cDNA library was made from the inbred line B734) was performedas described above using the following zap specific primers5′-AAGTCGACGCGGCCGCCACACCTAGTGCCAGGTCAG-3′ (SEQ ID No 17) and5′-ATCTCAATTGTACATTTCTCAGGA-3′ (SEQ ID No 18). A 450-bp PCR product wasobtained after PCR with zap-specific primers. Eight independent, becauseof their slight differences in lengths at their 5′ ends, 5′RACE PCRfragments generated with zap-specific primers were sequenced. In all thetranscripts from the LG2080 maize plants, there was an insertion ofadditional sequence in the coding region, which made the ZAP proteinlonger by 11 amino acids (980 amino acids, molecular mass ˜110.4 kDa).The Zn-finger I of ZAP2 was standard and read CKSCxxxHARC (FIG. 1B; SEQID No 11). The sequence difference may be due either to differencesbetween maize varieties, to the expression of two homologous genes, orto alternative splicing. In fact, maize may have at least two zap genes(FIG. 2B), which encode a transcript of 3.4-3.5 kb (FIG. 2D). The DNAgel blot experiment with a probe prepared from the zap cDNA showed thathomologous genes were present in Arabidopsis.

Structurally ZAP was very similar to PARP from animals. It had a wellconserved DNA-binding domain composed of two Zn-fingers (36% identityand 45% similarity to the DNA-binding domain of mouse PARP). Even higherhomology was shown by comparing only the sequences of the Zn-fingers,Ala¹-Phe¹⁶² in the mouse enzyme (44% identity and 54% similarity), or asubdomain downstream from the nuclear localization signal (NLS),Leu²³⁷-Ser³⁶⁰ in mouse PARP (40% identity and 50% similarity). Whereasthe bipartite nuclear localization signal characteristic of mammalianPARP could not be identified in ZAP, the sequence KRKK fitted amonopartite NLS (FIG. 1B). The putative automodification domain waspoorly conserved and was shorter in ZAP than in mouse PARP. Thecompilation of the homology of the catalytic domains between ZAP, NAP,APP and mouse PARP is shown in FIG. 2. It should be noted that theNAD⁺-binding domain of ZAP was more similar to the mammalian enzyme (48%identity) than to that of APP and NAP (40% and 42% sequence identity,respectively), whereas APP and NAP were 68% identical and 76% similar intheir catalytic domain.

Example 2 Demonstration that Non-Conventional PARP Protein has aDNA-Dependent Poly(ADP-Ribose) Polymerase Activity

APP is a DNA-Dependent Poly(ADP-ribose) Polymerase

A more detailed study of the APP protein (expressed in yeast) wasperformed to understand the activity of PARP-like proteins from the NAPclass. The choice of yeast as the organism for the expression andenzymatic analysis of the Arabidopsis APP protein was made for a numberof reasons. As an eukaryote, Saccharomyces cerevisiae is better suitedfor the expression of native proteins from other eukaryotic organisms,and unlike most other eukaryotic cells, it does not possess endogenousPARP activity (Lindahl et al., 1995).

The full-length app cDNA was placed in pYeDP1/8-2 under the control of agalactose-inducible yeast promoter in the following way the full-lengthapp cDNA was excised from pC3 (Lepiniec et al., 1995) as an XhoI-EcoRIfragment. The ends were filled in with the Klenow fragment of DNApolymerase 1, and the fragment was subcloned into the SmaI site of theyeast expression vector pYeDP1/8-2 (Cullin and Pompon, 1988). Theresulting expression vector pV8SPA (FIG. 4A) was transformed into S.cerevisiae strain DY.

For APP expression in E. coli, the complete coding region of the appcDNA was PCR amplified with Pfu DNA polymerase (Stratagene), using theprimers 5′-AGGATCCCATGGCGAACAAGCTCAAAGTGAC-3′ (SEQ ID No 19) and5′-AGGATCCTTAGTGCTTGTAGTTGAAT-3′ (SEQ ID No 20), and subcloned as aBamHI fragment into pET19b (Novagene, Madison, Wis.), resulting inpETSPA. The expression of the full-length APP in E. coli BL21 frompETSPA was very poor. To obtain better expression, pETSPA was digestedwith NcoI and NdeI or with SmaI, the ends were filled in by the Klenowfragment of DNA polymerase 1, and the plasmids were then self-ligated.Of the resulting plasmids pETΔNdeSPA and pETΔSmaSPA, only pETΔNdeSPAgave satisfactory expression of the truncated APP polypeptide (Met³¹⁰ toHis⁶³⁷) in E. coli BL21.

The expression of the APP in yeast was verified by Northern and Westernblot analysis. (FIG. 4) As the promoter in pV8SPA is inactive when cellsare grown on glucose and derepressed on galactose-containing media, theexpression was expected to be tightly regulated by the carbon source.However, Northern blot analysis of RNA and immunoblot analysis ofproteins in DY(pV8SPA) as compared to the control DY strain containingthe empty vector, showed that app mRNA and APP protein were expressed inyeast even when grown on glucose-containing media (FIG. 4B, lane 2). Thepeculiarity of the expression observed on glucose-containing medium wasthat both app mRNA and APP protein were shorter than the ones detectedafter induction with galactose (compare lanes 2 and 4 in FIG. 4B). TheAPP polypeptide with the higher molecular weight, (apparently afull-length protein) was only detected on galactose-containing medium,although such cells also expressed the truncated mRNA and protein. Themost probable explanation for this finding is that when the DY(pV8SPA)strain is grown on glucose, there is a leaky expression from theexpression cassette, with transcription beginning 200-300 bp downstreamfrom the transcription start observed after galactose induction. Thisshorter mRNA probably does not code for the first methionine (Met¹) ofAPP and, therefore, translation is initiated at Met⁷². This wouldexplain the observed difference of −5 kDa (calculated difference being7.5 kDa) in the molecular masses of the APP polypeptides from strainsgrown on glucose or on galactose. The possibility that the differencesin molecular masses may be attributed to self-modification throughpoly(ADP-ribos)ylation was ruled out by growing strains in the presenceof PARP inhibitors, such as 3ABA and nicotinamide (FIG. 4B, comparelanes 6 and 8 to lane 4).

To detect the synthesis of poly(ADP-ribose), total proteins wereextracted from yeast strains grown under different conditions andincubated in the presence of radioactively labeled NAD⁺. To preventsynthesis of poly(ADP-ribose) and possible automodification of the APPin vivo, strains were also grown in the presence of 3ABA, a reversibleinhibitor of PARP, which was subsequently removed from the proteinextracts during desalting. FIG. 5 shows that poly(ADP-ribose) issynthesized by protein extracts of DY(pV8SPA) grown on galactose (FIG.5A, lanes 1 and 2), but not by a strain containing the empty vector(FIG. 5A, lane 4). It can also be seen that Arabidopsis APP couldsynthesize polymers up to 40 residues in length (FIG. 5A, lane 1) withthe majority of the radioactivity being incorporated into 10-15-mer.This observation is consistent with the polymer sizes detected by otherauthors (Chen et al., 1994). More radioactivity was incorporated intopolymer when the yeast strain was grown with 3ABA than without (FIG. 5A,lane 1 compared to lane 2); the reason might be that either the APPextracted from inhibited cultures was less automodified (it is believedthat automodification inhibits the activity of PARP) or the labeled NAD⁺was used by the enzyme from the uninhibited culture for the extension ofexisting polymer, resulting in a lower specific activity overall. Underthe same reaction conditions poly(ADP-ribose) synthesized by human PARP,either in reaction buffer alone or in the presence of a yeast totalprotein extract from DY(pYeDP1/8-2) (FIG. 5A, lanes 5 and 6,respectively), showed much longer chains, possibly up to 400-mer (deMurcia and Ménissier de Murcia, 1994).

The stimulation of enzymatic activity by nicked DNA is a well knownproperty of PARP from animals (Alvarez-Gonzalez and Althaus, 1989). Wetherefore tested whether the activity of the APP protein was DNAdependent. After removal of yeast nucleic acids (DNA, RNA) and somebasic proteins from the galactose-grown DY(pV8SPA) protein extract thesynthesis of poly(ADP-ribose) was analyzed in the presence of increasingconcentrations of sonicated salmon sperm DNA. As can be seen in FIG. 5B,there was a direct correlation between the amount of DNA present in thereaction and the incorporation of ³²P-NAD⁺. Scanning of thephosphor-images indicated that ˜6-fold more radioactivity wasincorporated into poly(ADP-ribose) in the reaction mixture containing 40μg ml⁻ of DNA than into that with 2 μg ml⁻¹ of DNA (FIG. 5B, lanes 4 and2, respectively). The synthesis of the polymer was sensitive to 3ABA inthe reaction mix (FIG. 5B, lane 5).

APP is a Nuclear Protein

In animal cells PARP activity is localized in the nucleus (Schreiber etal., 1992). The intracellular localization, if nuclear, of APP couldprovide an important additional indication that APP is a bona fide plantPARP. To this end, the localization of the APP polypeptides in yeastcells was analyzed using anti-APP antisera. The APP polypeptidesynthesized in yeast grown on galactose was found mainly in the nucleus.This localization was unaffected by the presence in the media of thePARP inhibitors.

In addition, we tested whether APP was constitutively active in yeastcells, as has been reported for the human PARP (Collinge and Althaus,1994). Here, fixed yeast spheroplasts were incubated with monoclonal 10Hantibody, which specifically recognizes poly(ADP-ribose) polymers(Kawamitsu et al., 1984). A positive yellowish-green fluorescence signalwith 10H antibody was localized in the nucleus and was observed only inDY(pV8SPA) cells grown on galactose. Positive staining was greatlyreduced in cells grown in the presence of the PARP inhibitors, 3ABA andnicotinamide.

To identify the intracellular localization of APP in plant cells, awidely adopted approach in plant studies was used, i.e., the examinationof the subcellular location of a fusion protein formed between theprotein in question and a reporter gene, once the protein fusion wasproduced in transgenic plants or transfected cells (Citovsky et al.,1994; Sakamoto and Nagatani, 1996; Terzaghi et al, 1997; von Arnim andDeng, 1994). An N-terminal translational fusion of GUS with the part ofthe APP polypeptide extending from the Met¹ to Pro⁴⁰⁷ was made. Thetranslational fusion of APP with bacterial GUS was constructed asfollows. Plasmid pETSPA was cut with SmaI, treated with alkalinephosphatase, and ligated to a blunted NcoI-XbaI fragment from pGUS1(Plant Genetic Systems N.V., Gent, Belgium). The ligation mix wastransformed into E. coli XL-1 and cells were plated onto LB mediumsupplemented with 0.1 mM isopropyl-β-D-thiogalactopyranoside, 40 μg ml⁻¹5-bromo-4-chloro-3-indolyl-β-D-glucuronide, and 100 μg ml⁻ ofampicillin. In this way, pETSPAGUS was selected as blue colonies. Theexpression in E. coli of the ˜110-kDa fusion protein was confirmed by insitu GUS activity gels (Lee et al., 1995). The APP-GUS fusion was placedunder the control of the 35S promoter of the CaMV (the Klenow-bluntedBamHI fragment from pETSPAGUS was subcloned into SmaI-digested pJD330;Gallie and Walbot, 1992) and the resulting expression cassette wassubcloned as an XbaI fragment into the XbaI site of the pCGN1547 binaryvector (McBride and Summerfelt, 1990) to give pGCNSPAGUS. The pGCNSPAGUSwas finally introduced into A. tumefaciens C58C1 Rif^(R)(pGV2260) by thefreezing-thawing transformation procedure.

Expression of the fusion protein was verified in E. coli. The chimericcDNA under the control of the 35S CaMV promoter was stably integratedinto the tobacco genome. Progeny from four independent transgenictobacco plants were analyzed for the subcellular distribution of the GUSactivity after in situ histochemical staining (Jefferson et al., 1987).In 2-day-old seedlings GUS activity could be detected in cotyledons andin roots, but not in hypocotyls or root tips. Because of thetransparency of root tissues, GUS staining was clearly localized in thenuclei of root hairs and epidermal cells. Additionally, some diffuse,non-localized staining of other root cells was seen, in particular alongthe vascular cylinders. This non-nuclear GUS staining was morepronounced in leaf tissues. Whereas young true leaves or cotyledonsdisplayed intense blue staining of the nuclei, there was also somediffuse staining of the cytoplasm. In fully expanded leaves, however,GUS staining became homogenous and similar to the staining of controlplants transformed with GUS under the control of the CaMV 35S promoter,in which GUS was expressed in the cytoplasm. Eventually, older leaves orcotyledons exhibited practically no histochemically detectable GUSactivity, with the exception of the vascular bundles, where the GUSstaining could not be confined to any particular cell compartment.

Deficiency in DNA Ligase I Induces Expression of the App Gene

PARP in animal cells is one of the most abundant nuclear proteins andits activity is regulated by allosteric changes in the protein uponbinding to damaged DNA. We found that the app gene in Arabidopsis had arather low level of expression, suggesting that transcriptionalactivation of this gene might be essential for APP function in vivo. Totest this hypothesis, the expression of the app gene was studied duringin vivo genome destabilization caused by a DNA ligase I deficiency. AT-DNA insertion mutation, line SK1B2, in the Arabidopsis DNA ligase Igene was isolated previously (Babiychuk et al., 1997). The mutation islethal in the homozygous state, but the mutant allele shows normaltransmission through the gametes. We therefore expected that cellshomozygous for the mutation would die due to incomplete DNA synthesisduring the S phase of the cell cycle, soon after the fertilization ofthe mutant embryo sac with mutant pollen.

An app promoter-GUS translational fusion, in which the coding region ofGUS was fused in-frame with the first five amino acids of APP and 2 kbof app 5′ flanking sequences was constructed (SEQ ID No 21). The geneencoding the fusion protein was transformed into Arabidopsis. After twoback-crosses to a wild type, heterozygous plants transformed with apppromoter-GUS were crossed with Arabidopsis line SK1B2. The inflorescenceof the control plants and plants heterozygous for the ligase mutationwere stained for the activity of GUS. The GUS staining pattern mostlydetected in aging tissues probably reflects the expression of the appgene, although we have no firm evidence that all of the regulatorysequences were present in the constructs used. This pattern was the sameboth in the inflorescences of control plants, not carrying the mutantligase gene and plants heterozygous for a mutation. Approximatelyone-fourth of the ovules in the mutant plants with the fusion proteinare GUS positive. Closer microscopical examination showed that in theGUS-positive ovules only the gametophyte was stained. The onlydifference between the control plants and the mutant plant was amutation in a DNA ligase gene. We therefore conclude that the app geneis induced because of either the accumulation of DNA breaks, or thedeath of the mutant embryo sacs fertilized with mutant pollen. GUSstaining of embryo sacs was found to appear within 24 h afterpollination, or therefore very soon after fertilization.

Example 3 Construction of PCD Modulating Chimeric Genes and Introductionof the T-DNA Vectors Comprising such PCD Modulating Genes in anAgrobacterium Strain

3.1. Construction of the p35S:(dsRNA-APP) and p35S:(dsRNA-ZAP) Genes

Using standard recombinant DNA procedures, the following DNA regions areoperably linked, as schematically outlined in FIG. 6 (constructs 1 and5):

For the p35S:(dsRNA-ZAP) chimeric gene

-   -   a CaMV 35S promoter region (Odell et al., 1985)    -   a Cab22 leader region (Harpster et al., 1988)    -   a ZAP encoding DNA region (about complete) (the Arabidopsis        thaliana homologue to SEQ ID No 10, isolated by hybridization)    -   about 500 bp of the 5′ end of the ZAP2 encoding DNA region in        inverse orientation    -   a CaMV35S 3′ end region (Mogen et al., 1990)

For the p35S:(dsRNA-APP) chimeric gene

-   -   a CaMV 35S promoter region (Odell et al., 1985)    -   a Cab22 leader region (Harpster et al., 1988)    -   an APP encoding DNA region (about complete) (SEQ ID No 5)    -   about 500 bp of the 5′ end of the APP encoding DNA region in        inverse orientation    -   a CaMV35S 3′ end region (Mogen et al., 1990)        3.2. Construction of the pNOS:(dsRNA-APP) and PNOS:(dsRNA-ZAP)        Genes

Using standard recombinant DNA procedures, the following DNA regions areoperably linked, as schematically outlined in FIG. 6 (constructs 2 and6):

For the pNOS:(dsRNA-ZAP) chimeric gene

-   -   a NOS promoter region (Herrera-Estrella et al., 1983)    -   a Cab22 leader region (Harpster et al., 1988)    -   a ZAP encoding DNA region (about complete) (the Arabidopsis        thaliana homologue to SEQ ID No 10, isolated by hybridization)    -   about 500 bp of the 5′ end of the ZAP2 encoding DNA region in        inverse orientation    -   a CaMV35S 3′ end region (Mogen et al., 1990)

For the pNOS:(dsRNA-APP) chimeric gene

-   -   a NOS promoter region (Herrera-Estrella et al., 1983)    -   a Cab22 leader region (Harpster et al., 1988)    -   an APP encoding DNA region (about complete) (SEQ ID No 5)    -   about 500 bp of the 5′ end of the APP encoding DNA region in        inverse orientation    -   a CaMV35S 3′ end region (Mogen et al., 1990)        3.3. Construction of the pTA29:(dsRNA-APP) and PTA29:(dsRNA-ZAP)        Genes

Using standard recombinant DNA procedures, the following DNA regions areoperably linked, as schematically outlined in FIG. 6 (constructs 3 and7):

For the pTA29:(dsRNA-ZAP) chimeric gene

-   -   a TA29 promoter region (WO 89/10396)    -   a Cab22 leader region (Harpster et al., 1988)    -   a ZAP encoding DNA region (about complete) (the Arabidopsis        thaliana homologue to SEQ ID No 10, isolated by hybridization)    -   about 500 bp of the 5′ end of the ZAP2 encoding DNA region in        inverse orientation    -   a CaMV35S 3′ end region (Mogen et al., 1990)

For the pTA29:(dsRNA-APP) chimeric gene

-   -   a TA29 promoter region (WO 89/10396)    -   a Cab22 leader region (Harpster et al., 1988)    -   an APP encoding DNA region (about complete) (SEQ ID No 5)    -   about 500 bp of the 5′ end of the APP encoding DNA region in        inverse orientation    -   a CaMV35S 3′ end region (Mogen et al., 1990)        3.4. Construction of the DNTP303:(dsRNA-APP) and        DNTP303:(dsRNA-ZAP) Genes

Using standard recombinant DNA procedures, the following DNA regions areoperably linked, as schematically outlined in FIG. 6 (constructs 4 and8):

For the pNTP303:(dsRNA-ZAP) chimeric gene

-   -   a NTP303 promoter region (Wetering 1994)    -   a Cab22 leader region (Harpster et al., 1988)    -   a ZAP encoding DNA region (about complete) (the Arabidopsis        thaliana homologue to SEQ ID No 10, isolated by hybridization)    -   about 500 bp of the 5′ end of the ZAP2 encoding DNA region in        inverse orientation    -   a CaMV35S 3′ end region (Mogen et al., 1990)

For the pNTP303:(dsRNA-APP) chimeric gene

-   -   a NTP303 promoter region (Wetering, 1994)    -   a Cab22 leader region (Harpster et al., 1988)    -   an APP encoding DNA region (about complete) (SEQ ID No 5)    -   about 500 bp of the 5′ end of the APP encoding DNA region in        inverse orientation    -   a CaMV35S 3′ end region (Mogen et al., 1990)        3.5 Construction of the Chimeric Marker Genes

Using standard recombinant DNA procedures, the following DNA regions areoperably linked, as schematically outlined in FIG. 6:

For the gat marker gene

-   -   an Act2 promoter region (An et al., 1996)    -   a aminoglycoside 6′-acetyltransferase encoding DNA (WO 94/26913)    -   a 3′ end region of a nopaline synthase gene (Depicker et al.,        1982)

For the bar marker gene

-   -   an Act2 promoter region (An et al., 1996)    -   a phosphinotricin acetyltransferase encoding DNA (U.S. Pat. No.        5,646,024)    -   a 3′ end region of a nopaline synthase gene (Depicker et al.,        1982)        3.6. Construction of the T-DNA Vectors Comprising the PCD        Modulating Chimeric Genes

Using appropriate restriction enzymes, the chimeric PCD modulating genesdescribed under 3.1 to 3.5 are excised and introduced in the polylinkerbetween the T-DNA borders of a T-DNA vector derived from pGSV5 (WO97/13865) together with either the gat marker gene or the bar markergene. The resulting T-DNA vectors are schematically represented in FIG.6.

3.7. Introduction of the T-DNA Vectors in Agrobacterium

The T-DNA vectors are introduced in Agrobacterium tumefaciensC58C1Rif(pGV4000) by electroporation as described by Walkerpeach andVelten (1995) and transformants are selected using spectinomycin andstreptomycin.

Example 4 Agrobacterium-Mediated Transformation of Arabidopsis thalianawith the T-DNA Vectors of Example 3

The Agrobacterium strains are used to transform Arabidopsis thalianavar. C24 applying the root transformation method as described byValvekens et al. (1992). The explants are coinfected with theAgrobacteria strains containing the dsRNA-APP respectively the dsRNA-ZAPconstructs. The dsRNA-APP constructs are used in combination with thepact:bargene. The dsRNA-ZAP constructs are used in combination with thepact-gat gene. Transformants are selected for phosphinothricinresistance. The regenerated rooted transgenic lines are tested for thepresence of the other T-DNA by screening for kanamycin resistance.Transgenic lines containing both T-DNA's are transferred to thegreenhouse. The phenotype of the T0-transgenic lines is scored and theT1-generations are studied further in more detail.

Example 5 Agrobacterium-Mediated Transformation of Brassica napus withthe T-DNA Vectors of Example 3

The Agrobacterium strains are used to transform the Brassica napus var.N90-740 applying the hypocotyl transformation method essentially asdescribed by De Block et al. (1989), except for the followingmodifications:

-   -   hypocotyl explants are precultured for 1 day on A2 medium [MS,        0.5 g/l Mes (pH5.7), 1.2% glucose, 0.5% agarose, 1 mg/l 2,4-D,        0.25 mg/l naphthalene acetic acid (NAA) and 1 mg/l        6-benzylaminopurine (BAP)].    -   infection medium A3 is MS, 0.5 g/l Mes (pH5.7), 1.2% glucose,        0.1 mg/l NM, 0.75 mg/l BAP and 0.01 mg/l gibberellinic acid        (GA3).    -   selection medium A5G is MS, 0.5 g/l Mes (pH5.7), 1.2% glucose,        40 mg/l adenine. SO₄, 0.5 g/l polyvinylpyrrolidone (PVP), 0.5%        agarose, 0.1 mg/l NM, 0.75 mg/l BAP, 0.01 mg/l GA3, 250 mg/l        carbenicillin, 250 mg/l triacillin, 5 mg/l AgNO₃ for three        weeks. After this period selection is continued on A5J medium        (similar a A5G but with 3% sucrose)    -   regeneration medium A6 is MS, 0.5 g/l Mes (pH5.7), 2% sucrose,        40 mg/l adenine. SO₄, 0.5 g/l PVP, 0.5% agarose, 0.0025 mg/l BAP        and 250 mg/l triacillin.    -   healthy shoots are transferred to rooting medium which was A9:        half concentrated MS, 1.5% sucrose (pH5.8), 100 mg/l triacillin,        0.6% agar in 1 liter vessels.        MS stands for Murashige and Skoog medium (Murashige and Skoog,        1962)

For introducing both the dsRNA-APP and the dsRNA-ZAP T-DNA constructsinto a same plant cell the co-transformation method is applied,essentially as described by De Block and Debrouwer (1991). Transformedplant lines are selected on phosphinothricin containing medium afterwhich the presence of the second T-DNA is screened by testing theregenerated rooted shoots for kanamycin resistance. In theco-transformation experiments, the dsRNA-APP constructs are used incombination with the pact:bar gene. The dsRNA-ZAP constructs are used incombination with the pact:gat gene. Transgenic lines containing bothT-DNA's are transferred to the greenhouse. The phenotype of theT0-transgenic lines is scored and the T1-generations are studied furtherin more detail.

Example 6 In Vitro Assay to Test Vigor of Plant Lines

6.1. Fitness Assay for Brassica napus

Media and Reaction Buffers

-   -   Sowing medium:        -   Half concentrated Murashige and Skoog salts        -   2% sucrose        -   pH 5.8        -   0.6% agar    -   Callus inducing medium: A2S        -   MS medium, 0.5 g/l Mes (pH 5.8), 3% sucrose, 40 mg/l            adenine-SO₄, 0.5% agarose, 1 mg/l 2,4-D, 0.25 mg/l NM, 1            mg/l BAP    -   Incubation medium:        -   25 mM K-phosphate buffer pH5.8        -   2% sucrose        -   1 drop Tween20 for 25 ml medium    -   Reaction buffer:        -   50 mM K-phosphate buffer pH7.4        -   10 mM 2,3,5-triphenyltetrazoliumchloride (TTC) (=3.35 mg/ml)        -   1 drop Tween20 for 25 ml buffer            Sterilization of Seeds and Growing of the Seedlings    -   Seeds are soaked in 70% ethanol for 2 min, then        surface-sterilized for 15 min in a sodium hypochlorite solution        (with about 6% active chlorine) containing 0.1% Tween20.        Finally, the seeds are rinsed with 1 l of sterile distilled        water. Put 7 seeds/1 l vessel (Weck) containing about 75 ml of        sowing medium. The seeds are germinated at 23° C. and 30        μEinstein/s⁻¹m⁻² with a daylength of 16 h.    -   The line N90-740 is always included for standardization between        experiments.        Preculture of the Hypocotyl Explants    -   12-14 days after sowing, the hypocotyls are cut in about 7 mm        segments. 25 hypocotyls/Optilux Petridisch (Falcon S1005)    -   The hypocotyl explants are cultured for 4 days on medium A2S at        23-25° C. (at 30 μEinstein/s⁻¹m⁻²).        -   □P.S.: about 150-300 hypocotyl explants/line are needed to            carry out the assay    -   Transfer the hypocotyl explants to Optilux Petridishes (Falcon        S1005) containing 30 ml of incubation medium.    -   Incubate for about 20 hours at 24° C. in the dark.        TTC-Assay    -   Transfer 150 hypocotyl explants to a 50 ml Falcon tube.    -   Wash with reaction buffer (without TTC).    -   Add 25 ml-30 ml of reaction buffer/tube.        -   tube 1□ no TTC added            -   for measuring background absorption            -   one line/experiment is sufficient        -   tube 2□+10 mM TTC        -   (explants have to be submerged, but do not vacuum            infiltrate!)    -   turn tubes upside down    -   Incubate for about 1 hour in the dark at 26° C. (no end        reaction!)    -   Wash hypocotyls with deionized water    -   Remove water    -   Freeze at −70° C. for 30 min.    -   Thaw at room°t (in the dark)    -   Add 50 ml ethanol (technical)    -   Extract reduced TTC-H by shaking for 1 hour    -   Measure absorptions of extracts at 485 nm        -   P.S.: reduced UTC-His not stable □ keep in the dark and            measure O.D.₄₈₅ as soon as possible            O.D._(485(TTC-H))=(O.D.₄₈₅+TTC)−(O.D.₄₈₅−TTC)    -   Comparison of the TTC-reducing capacities between samples of        different independent experiments can be done by setting the        TTC-reducing capacity of N90-740 in the different experiment at        100%.    -   Lines with a high TTC-reducing capacity are vigorous, while        lines with a low TTC-reducing capacity are weak.        6.2. Fitness Assay Arabidopsis        Media and Reaction Buffers    -   Plant medium: Half concentrated Murashige and Skoog salts        -   1.5% sucrose        -   pH 5.8        -   0.6% agar        -   →autoclave 15 min.        -   add filter sterilized—100 mg/l myo-inositol            -   0.5 mg/l pyridoxine            -   0.5 mg/l nicotinic acid            -   1 mg/l thiamine    -   Incubation medium: 10 mM K-phosphate buffer pH5.8        -   2% sucrose        -   1 drop Tween20 for 25 ml medium    -   Reaction buffer: 50 mM K-phosphate buffer pH7.4        -   10 mM 2,3,5-triphenyltetrazoliumchloride (TTC) (=3.35 mg/ml)        -   1 drop Tween20 for 25 ml buffer            Arabidopsis Plants    -   Sterilization of Arabidopsis Seeds        -   2 min. 70% ethanol        -   10 min. bleach (6% active chlorine)+1 drop Tween 20 for 20            ml solution        -   wash 5 times with sterile water            -   P.S.: sterilization is done in 2 ml eppendorf tubes                Arabidopsis seeds sink to the bottom of the tube,                allowing removal of the liquids by means of a 1 ml                pipetman    -   Growing of Arabidopsis Plants        -   Seeds are sown in ‘Intergrid Tissue Culture disks of Falcon’            (nr. 3025) containing ±100 ml of plant medium: 1 seed/grid.        -   Plants are grown at 23° C.            -   40 μEinstein s⁻¹m⁻²            -   16 hours light-8 hours dark        -   for about 3 weeks (plants start to form flower buds)        -   →P.S.: *about 90-110 plants/line are needed to carry out the            assay            -   include control line (C24; Columbia; . . . ) for                calibration                Pre-Incubation    -   Harvest Arabidopsis shoots by cutting of roots (by means of        scissors)    -   Put each shoot immediately in incubation medium (shoots have to        be submerged, but do not vacuum infiltrate)    -   Incubation medium: 1150 ml in ‘Intergrid Tissue Culture disks of        Falcon’ (nr. 3025)        -   a) incubation medium: for quantification of background            absorption (see TTC-assay)        -   b) incubation medium        -   c) incubation medium+2 mM niacinamide    -   30-35 shoots/petridish (but same amount of shoots for all lines        and for each condition)    -   Incubate at 24° C. in the dark for ±20 hours        TTC-Assay    -   Transfer shoots to 50 ml Falcon tubes    -   Wash with reaction buffer (without TTC)    -   Add 30-35 ml of reaction buffer/tube        -   a) no TTC added (for measuring background absorption)        -   b and c)+10 mM TTC        -   (Shoots have to be submerged, but do not vacuum infiltrate!)    -   Incubate for about 2 hours in the dark at 26° C. (no end        reaction!)    -   Wash shoots with deionized water    -   Remove water    -   Freeze at −70° C. for 30 min.    -   Thaw at room°t (in the dark)    -   Add 50 ml ethanol (technical)    -   Extract reduced TTC-H by shaking for 1 hour    -   Measure absorptions of extracts at 485 nm        -   P.S.: reduced TTC-His not stable e keep in the dark and            measure O.D.₄₈₅ as soon as possible    -   Compare reducing profiles of tested lines versus control line        (for population of 30 to 35 plants)        O.D._(485(TTC-H))=(O.D.₄₈₅+TTC)−(O.D.₄₈₅−TTC)    -   Comparison of the TTC-reducing capacities between samples of        different independent experiments can be done by setting the        TTC-reducing capacity of control line (C24; Columbia; . . . ) in        the different experiments at 100%.    -   Lines with a high TTC-reducing capacity are vigorous, while        lines with a low TTC-reducing capacity are weak.    -   If the addition of niacinamide to the incubation medium results        in a higher TTC-reducing capacity indicates to a lower fitness        (as shown for C24 and Columbia).

Example 7 Phenotypic Analyses of the Transgenic Lines Containing BothdsRNA-APP and dsRNA-ZAP Constructs

The flower phenotype and pollen viability (Alexander staining(Alexander, 1969) and germination assay) of the T0-lines containingdsRNA-APP and dsRNA-ZAP under the control of tapetum or pollen specificpromoters were scored. For Arabidopsis, the T1-generation is obtained byselving or if the plants are male sterile by backcrossing using pollenof non-transformed wild type plants. For Brassica napus, theT1-generation is always obtained by backcrossing using pollen ofnon-transformed plants.

T1-seed is germinated on kanamycin containing medium after which theresistant plants are scored by means of the ammonium-multiwell assay forphosphinothricine resistance (De Block et al., 1995). One half of theplants that contains both T-DNA's is transferred to the greenhouse toscore the male fertility of the plants, while the other half is used toquantify the vigor of the plants by means of the fitness assay. Forplants comprising combinations (APP/ZAP) of PCD modulating genes undercontrol of 35S or NOS promoter, a high vigor is observed in a number ofthe transgenic lines.

For plants comprising combinations (APP/ZAP) of PCD modulating genesunder control of TA29 male sterility is observed in a number of thetransgenic lines.

For plants comprising combinations (APP/ZAP) of PCD modulating genesunder control of NTP303 sterile pollen is observed in a number thetransgenic lines.

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1. A method for obtaining a cell of a plant with high vigor, whencompared to a control plant cell, comprising the steps of: i)identifying an endogenous PARP encoding gene or cDNA from said plantcomprising: a. performing PCR on genomic or cDNA from said plant at anannealing temperature of at least 45° C. using a primer pair selectedfrom the nucleotide sequences comprising at least 20 consecutivenucleotides of the nucleotide sequence of SEQ ID No.: 1, SEQ ID No.: 3,SEQ ID NO.: 5 or SEQ ID No.: 10; b. performing PCR on genomic or cDNAfrom said plant at an annealing temperature of at least 45° C. using aprimer pair selected from the nucleotide sequences comprising at least20 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 1from nucleotide position 2558 to 2704, the sequence of SEQ ID NO: 3 fromnucleotide position 1595 to 1747, the sequence of SEQ ID NO: 5 fromnucleotide position 1575 to 1724, or the sequence of SEQ ID NO: 10 fromnucleotide position 2559 to 2705; or c. performing PCR on genomic orcDNA from said plant at an annealing temperature of at least 45° C.using a primer pair selected from the following nucleotide sequences:SEQ ID No.: 13 and SEQ ID No.: 14; SEQ ID No.: 15 and SEQ ID No.: 16;SEQ ID No.: 17 and SEQ ID No.: 18; SEQ ID No.: 19 and SEQ ID No.: 20;and ii) introducing a chimeric gene to said cell to yield a transgeniccell, wherein said chimeric gene comprises the following operably linkedDNA regions: a) a plant-expressible promoter; b) a DNA region, whichwhen transcribed yields an RNA molecule, capable of reducing theexpression of said endogenous PARP encoding gene or cDNA; and c) a DNAregion involved in transcription termination and polyadenylation whereinsaid RNA molecule for introduction into said cell of said plantcomprises i) a sense nucleotide sequence comprising at least 100consecutive nucleotides from said endogenous PARP encoding gene or cDNAof said plant; and ii) an antisense nucleotide sequence comprising atleast 100 consecutive nucleotides from the complement of said sensenucleotide sequence; said sense and said antisense nucleotide sequencebeing capable of combining into a double stranded RNA region; andwherein said vigor of said cell of said plant can be measured bymeasuring the capacity of explants of said plant to reduce2,3,5-triphenyltetrazoliumchloride.
 2. The method of claim 1, whereinsaid PARP encoding gene or cDNA is of the ZAP class.
 3. The method ofclaim 1, wherein said PARP encoding gene or cDNA is of the NAP class. 4.The method of claim 1, wherein said plant expressible promoter is aconstitutive promoter.
 5. The method of claim 1, further comprising thestep of regenerating a transgenic plant from said transgenic plant cell.6. The method of claim 5, further comprising the step of producing moreplants comprising said chimeric gene by a conventional breeding scheme.7. The method of claim 1, wherein said sense nucleotide sequencecomprises at least 100 consecutive nucleotides selected from thesequence of SEQ ID NO: 1 from nucleotide position 113 to 1189, thesequence of SEQ ID NO: 3 from nucleotide position 107 to 583, thesequence of SEQ ID NO: 5 from nucleotide position 131 to 542, or thesequence of SEQ ID NO: 10 from nucleotide position 81 to
 1180. 8. Themethod of claim 1, wherein said sense nucleotide sequence comprises atleast 100 consecutive nucleotides from the sequence of SEQ ID NO: 1 fromnucleotide position 2558 to 2704, the sequence of SEQ ID NO: 3 fromnucleotide position 1595 to 1747, the sequence of SEQ ID NO: 5 fromnucleotide position 1575 to 1724, or the sequence of SEQ ID NO: 10 fromnucleotide position 2559 to
 2705. 9. The method of claim 1, wherein saidRNA molecule comprises a sense nucleotide sequence comprising at least250 consecutive nucleotides from SEQ ID NO: 1, 3, 5, or
 10. 10. Themethod of claim 1, wherein said RNA molecule comprises a sensenucleotide sequence comprising at least 500 consecutive nucleotides fromSEQ ID NO: 1, 3, 5, or
 10. 11. A Zea mays, Brassica napus, orArabidopsis thaliana plant exhibiting high vigor when compared to acontrol plant obtained by the method of claim 5, said plant comprising achimeric gene comprising the following operably linked DNA regions: a) aplant-expressible promoter; b) a DNA region, which when transcribedyields an RNA molecule capable of reducing the expression of saidendogenous PARP encoding gene or cDNA; and c) a DNA region involved intranscription termination and polyadenylation; wherein said RNA moleculetranscribed from said chimeric gene in said plant comprises i) a sensenucleotide sequence comprising at least 100 consecutive nucleotides fromsaid endogenous PARP encoding gene or cDNA of said plant; and ii) anantisense nucleotide sequence comprising at least 100 consecutivenucleotides from the complement of said sense nucleotide sequence; saidsense and antisense nucleotide sequence being capable of combining intoa double stranded RNA region; and wherein said vigor of said cell ofsaid plant can be measured by measuring the capacity of explants of saidplant to reduce 2,3,5-triphenyltetrazoliumchloride.
 12. The transgenicplant of claim 11, wherein said PARP encoding gene or cDNA is of the ZAPclass.
 13. The transgenic plant of claim 11, wherein said PARP encodinggene or cDNA is of the NAP class.
 14. The transgenic plant of claim 11,wherein said plant expressible promoter is a constitutive promoter. 15.The transgenic plant of claim 11, wherein said sense nucleotide sequencecomprises at least 100 consecutive nucleotides from the sequence of SEQID NO: 1 from nucleotide position 113 to 1189, the sequence of SEQ IDNO: 3 from nucleotide position 107 to 583, the sequence of SEQ ID NO: 5from nucleotide position 131 to 542, or the sequence of SEQ ID NO: 10from nucleotide position 81 to
 1180. 16. The transgenic plant of claim11, wherein said sense nucleotide sequence comprises at least 100consecutive nucleotides from the sequence of SEQ ID NO: 1 fromnucleotide position 2558 to 2704, the sequence of SEQ ID NO: 3 fromnucleotide position 1595 to 1747, the sequence of SEQ ID NO: 5 fromnucleotide position 1575 to 1724, or the sequence of SEQ ID NO: 10 fromnucleotide position 2559 to
 2705. 17. The transgenic plant of claim 11,wherein said RNA molecule comprises a sense nucleotide sequencecomprising of at least 250 consecutive nucleotides from SEQ ID NO: 1, 3,5, or
 10. 18. The transgenic plant of claim 11, wherein said RNAmolecule comprises a sense nucleotide sequence comprising at least 500consecutive nucleotides from SEQ ID NO: 1, 3, 5, or
 10. 19. A seed ofthe plant of claim 11, comprising said chimeric gene.
 20. The chimericgene described in claim
 1. 21. The chimeric gene of claim 20, whereinsaid PARP encoding gene or cDNA is of the ZAP class.
 22. The chimericgene of claim 20, wherein said PARP encoding gene or cDNA is of the NAPclass.
 23. The chimeric gene of claim 20, wherein said plant expressiblepromoter is a constitutive promoter.
 24. The chimeric gene of claim 20,wherein said sense nucleotide sequence comprises at least 100consecutive nucleotides from the sequence of SEQ ID NO: 1 fromnucleotide position 113 to 1189, the sequence of SEQ ID NO: 3 fromnucleotide position 107 to 583, the sequence of SEQ ID NO: 5 fromnucleotide position 131 to 542, or the sequence of SEQ ID NO: 10 fromnucleotide position 81 to
 1180. 25. The chimeric gene of claim 20,wherein said sense nucleotide sequence comprises at least 100consecutive nucleotides selected from the sequence of SEQ ID NO: 1 fromnucleotide position 2558 to 2704, the sequence of SEQ ID NO: 3 fromnucleotide position 1595 to 1747, the sequence of SEQ ID NO: 5 fromnucleotide position 1575 to 1724, or the sequence of SEQ ID NO: 10 fromnucleotide position 2559 to
 2705. 26. The chimeric gene of claim 20,wherein said RNA molecule comprises a sense nucleotide sequencecomprising at least 250 consecutive nucleotides from SEQ ID NO: 1, 3, 5,or
 10. 27. The chimeric gene of claim 20, wherein said RNA moleculecomprises a sense nucleotide sequence comprising at least 500consecutive nucleotides from SEQ ID NO: 1, 3, 5, or
 10. 28. A method forobtaining a cell of a plant with high vigor, when compared to a controlplant cell, comprising the steps of: i) identifying an endogenous PARPencoding gene or cDNA from said plant comprising: a. performing ahybridization reaction on genomic or cDNA from said plant using a probecomprising the nucleotide sequence of SEQ ID No.: 1, SEQ ID No.: 3, SEQID No.: 5, or SEQ ID No.: 10; b. performing a hybridization reaction ongenomic or cDNA from said plant using a probe comprising at least 20consecutive nucleotides of a nucleotide sequence of SEQ ID No.: 1, SEQID No.: 3, SEQ ID No.: 5, or SEQ ID No.: 10; c. performing ahybridization reaction on genomic or cDNA from said plant using a probecomprising the nucleotide sequence of SEQ ID NO.: 1 from nucleotideposition 113 to 1189, the sequence of SEQ ID NO.: 3 from nucleotideposition 107 to 583, the sequence of SEQ ID NO.: 5 from nucleotideposition 131 to 542, or the sequence of SEQ ID NO.: 10 from nucleotideposition 81 to 1180; or d. performing a hybridization reaction ongenomic or cDNA from said plant using a probe comprising the nucleotidesequence of SEQ ID NO: 1 from nucleotide position 2558 to 2704, thesequence of SEQ ID NO: 3 from nucleotide position 1595 to 1747, thesequence of SEQ ID NO: 5 from nucleotide position 1575 to 1724, or thesequence of SEQ ID NO: 10 from nucleotide position 2559 to 2705; and ii)introducing a chimeric gene to said cell to yield a transgenic cell,wherein said chimeric gene comprises the following operably linked DNAregions: a) a plant-expressible promoter; b) a DNA region, which whentranscribed yields an RNA molecule, capable of reducing the expressionof said endogenous PARP encoding gene or cDNA; and c) a DNA regioninvolved in transcription termination and polyadenylation wherein saidRNA molecule for introduction into said cell of said plant comprises i)a sense nucleotide sequence comprising at least 100 consecutivenucleotides from said endogenous PARP encoding gene or cDNA of saidplant; and ii) an antisense nucleotide sequence comprising at least 100consecutive nucleotides from the complement of said sense nucleotidesequence; said sense and said antisense nucleotide sequence beingcapable of combining into a double stranded RNA region; and wherein saidvigor of said cell of said plant can be measured by measuring thecapacity of explants of said plant to reduce2,3,5-triphenyltetrazoliumchloride.
 29. The method of claim 28, whereinthe step of identifying an endogenous PARP encoding gene or cDNA fromsaid plant comprises hybridizing said gene or cDNA to a probe comprisingthe nucleotide sequence of SEQ ID No.: 1, SEQ ID No.: 3, SEQ ID No.: 5,or SEQ ID No.:
 10. 30. The method of claim 28, wherein the step ofidentifying an endogenous PARP encoding gene or cDNA from said plantcomprises hybridizing said gene or cDNA to a probe comprising at least20 consecutive nucleotides of a nucleotide sequence of SEQ ID No.: 1,SEQ ID No.: 3, SEQ ID No.: 5, or SEQ ID No.:
 10. 31. The method of claim28, wherein the step of identifying an endogenous PARP encoding gene orcDNA from said plant comprises hybridizing said gene or cDNA to a probecomprising the nucleotide sequence of SEQ ID No.: 1 from nucleotideposition 113 to 1189, the sequence of SEQ ID No.: 3 from nucleotideposition 107 to 583, the sequence of SEQ ID No.: 5 from nucleotidesequence position 131 to 542, or the sequence of SEQ ID No.: 10 fromnucleotide position 81 to
 1180. 32. The method of claim 1, wherein thestep of identifying an endogenous PARP encoding gene or cDNA from saidplant comprises performing PCR at an annealing temperature of at least45° C. using a primer pair selected from the nucleotide sequencescomprising at least 20 consecutive nucleotides of the nucleotidesequence of SEQ ID No.: 1, SEQ ID No.: 3, SEQ ID No.: 5, or SEQ ID No.:10.
 33. The method of claim 1, wherein the step of identifying anendogenous PARP encoding gene or cDNA from said plant comprisesperforming PCR at an annealing temperature of at least 45° C. using aprimer pair selected from the following nucleotide sequences: SEQ IDNo.: 13 and SEQ ID No.: 14; SEQ ID No.: 15 and SEQ ID No.: 16; SEQ IDNo.: 17 and SEQ ID No.: 18; SEQ ID No.: 19 or and SEQ ID No.:
 20. 34.The method of claim 1, wherein the step of identifying an endogenousPARP encoding gene or cDNA from said plant comprises performing PCR atan annealing temperature of at least 45° C. using a primer pair selectedfrom the nucleotide sequences comprising at least 20 consecutivenucleotides of the nucleotide sequence of SEQ ID NO: 1 from nucleotideposition 2558 to 2704, the sequence of SEQ ID NO: 3 from nucleotideposition 1595 to 1747, the sequence of SEQ ID NO: 5 from nucleotideposition 1575 to 1724, or the sequence of SEQ ID NO: 10 from nucleotideposition 2559 to
 2705. 35. The method of claim 28, wherein the step ofidentifying an endogenous PARP encoding gene or cDNA from said plantcomprises hybridizing said gene or cDNA to a probe comprising thenucleotide sequence of SEQ ID NO: 1 from nucleotide position 2558 to2704, the sequence of SEQ ID NO: 3 from nucleotide position 1595 to1747, the sequence of SEQ ID NO: 5 from nucleotide position 1575 to1724, or the sequence of SEQ ID NO: 10 from nucleotide position 2559 to2705.